Multiplex gene removal by two-step polymerase chain reactions

Precise DNA manipulation is critical for molecular biotechnology. Restriction enzyme-based approaches are limited by their requirement of specific enzyme sites. Restriction-free cloning has greatly improved the flexibility and speed of precise DNA assembly. Most of these approaches focus on DNA assembly rather than gene removal. Here we present a polymerase chain reaction (PCR)-based cloning method that allows removal of multiple gene segments from plasmids without using restriction enzymes and thermostable ligase. We demonstrate simultaneous removal of three gene segments from a plasmid. This approach could be beneficial to DNA library construction, genetic and protein engineering, and synthetic biology.     

Regulation of apoptosis by E3 ubiquitin ligases in ubiquitin proteasome system

Apoptosis is an organised ATP‐dependent programmed cell death that organisms have evolved to maintain homoeostatic cell numbers and eliminate unnecessary or unhealthy cells from the system. Dysregulation of apoptosis can have serious manifestations culminating into various diseases, especially cancer. Accurate control of apoptosis requires regulation of a wide range of growth enhancing as well as anti‐oncogenic factors. Appropriate regulation of magnitude and temporal expression of key proteins is vital to maintain functional apoptotic signalling. Controlled protein turnover is thus critical to the unhindered operation of the apoptotic machinery, disruption of which can have severe consequences, foremost being oncogenic transformation of cells. The ubiquitin proteasome system (UPS) is one such major cellular pathway that maintains homoeostatic protein levels. Recent studies have found interesting links between these two fundamental cellular processes, wherein UPS depending on the cue can either inhibit or promote apoptosis. A diverse range of E3 ligases are involved in regulating the turnover of key proteins of the apoptotic pathway. This review summarises an overview of key E3 ubiquitin ligases involved in the regulation of the fundamental proteins involved in apoptosis, linking UPS to apoptosis and attempts to emphasize the significance of this relationship in context of cancer.     

An Engineered Membrane to Measure Electroporation: Effect of Tethers and Bioelectronic Interface

This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.     

Enzymic basis of deranged foetal flavin-nucleotide metabolism consequent on immunoneutralization of maternal riboflavin carrier protein in the pregnant rat.

A comparison of the kinetic and other parameters of enzymes of flavin-nucleotide metabolism in the whole foetus vis-à-vis the maternal liver in the pregnant rat revealed relatively lower activities of foetal flavokinase and FAD pyrophosphorylase. Passive immunoneutralization of the maternal riboflavin carrier protein suppresses foetal FAD pyrophosphorylase rather selectively. Additionally, although the activities of foetal nucleotide pyrophosphatase and FMN phosphatase were unchanged owing to immunoneutralization, higher activities of these enzymes in the whole foetus as compared with the maternal liver may be responsible for the drastic depletion of FAD levels that precipitates foetal degeneration.     

Characterization of cysteine thiol modifications based on protein microenvironments and local secondary structures

We have demonstrated earlier that protein microenvironments were conserved around disulfide‐bridged cystine motifs with similar functions, irrespective of diversity in protein sequences. Here, cysteine thiol modifications were characterized based on protein microenvironments, secondary structures and specific protein functions. Protein microenvironment around an amino acid was defined as the summation of hydrophobic contributions from the surrounding protein fragments and the solvent molecules present within its first contact shell. Cysteine functions (modifications) were grouped into enzymatic and non‐enzymatic classes. Modifications studied were—disulfide formation, thio‐ether formation, metal‐binding, nitrosylation, acylation, selenylation, glutathionylation, sulfenylation, and ribosylation. 1079 enzymatic proteins were reported from high‐resolution crystal structures. Protein microenvironments around cysteine thiol, derived from above crystal structures, were clustered into 3 groups—buried‐hydrophobic, intermediate and exposed‐hydrophilic clusters. Characterization of cysteine functions were statistically meaningful for 4 modifications (disulfide formation, thioether formation, sulfenylation, and iron/zinc binding) those have sufficient amount of data in the current dataset. Results showed that protein microenvironment, secondary structure and protein functions were conserved for enzymatic cysteine functions, in contrast to the same function from non‐enzymatic cysteines. Disulfide forming enzymatic cysteines were tightly packed within intermediate protein microenvironment cluster, have alpha‐helical conformation and mostly belonged to CxxC motif of electron transport proteins. Disulfide forming non‐enzymatic cysteines did not belong to conserved motif and have variable secondary structures. Similarly, enzymatic thioether forming cysteines have conserved microenvironment compared to non‐enzymatic cystienes. Based on the compatibility between protein microenvironment and cysteine modifications, more efficient drug molecules could be designed against cysteine‐related diseases.     

Genotype-Dependent morphogenetic potentiality of various explants of a food legume,the pigeon pea (Cajanus cajan L.)

Summary The morphogenetic response of various explants of seven different cultivars of a food legume, the pigeon pea (Cajanus cajan L.), has been studied. The stimulation and elongation of shoot buds into shoots derived from the mature embryo axis and intact seed on Murashige and Skoog’s medium supplemented with 2.32µM kinetin and 22.2µM benzyladenine was found to be optimum in Murashige and Skoog’s medium supplemented with 0.46µM kinetin, 0.53µM naphthalene acetic acid, and 0.29µM gibberellic acid. Even though the response of these two explants for formation of shoot buds in all the genotypes is 30–100% depending on media composition, subsequent growth and elongation of these shoot buds into plants is genotype dependent and is restricted to two genotypes. Cotyledon and epicotyl explants of pigeon pea cultivars on the other hand differentiated directly into four to eight and two to four shoots, respectively, depending on the media composition and genotype. In vitro rhizogenesis of regenerated shoots was 80% and the survival of these plantlets in the field was 70–80%. NCL Communication no.: 5667.     

In vitro plant regeneration from leaf callus inPiper colubrinum Link

Callus-mediated shoot regeneration from leaf explants ofPhytophthora resistant pepper (Piper colubrinum Link.) is described. The effect of basal media composition and growth regulators onin vitro response of explants was evaluated. Shoot buds were induced and elongated on half-strength MS medium containing 2.0 mg l^–1 BA and 0.5 mg l^–1 NAA , as well as 1.0 mg l^–1 BA and 0.5 mg l^–1 2,4-D. The shoots were rooted in half-strength MS medium with or without IAA or IBA, and then were transferred to soil with 100% survival.     

Identification and expression profiling of a new β-amyrin synthase gene (<Emphasis Type="Italic">GmBAS3</Emphasis>) from soybean

Cyclization of 2,3-oxidosqualene by different oxidosqualene cyclase (OSC) genes is responsible for sapogenin heterogeneity. The very first phase is the conversion of 2,3-oxidosqualene into β-amyrin by β-amyrin synthase (BAS) gene, a member of OSC family, in soy saponin biosynthesis pathway. This paper reports the identification of a new BAS gene (GmBAS3) and its expression pattern in soybean (Glycine max (L.) Merr.). GmBAS3 gene was identified by PCR/RACE method with an open reading frame of 2286 bp nucleotides encoding a 762 amino acid long protein devouring a characteristic QW motif repeated five times and DCTAE motif. GmBAS3 shared 96 and 92% homology with Glycyrrhiza uralensis BAS and Lotus japonicus putative BAS respectively. Expression of the gene was detected by RT-PCR in regard to seedlings age and tissue type. A spatio-temporal expression of GmBAS3 was found in 21-day-old seedlings in the hypocotyls, young leaves and mature leaves but not observed in stem and root tissues. No expression was perceived in 10-day-old seedling. This study also support the premise that β-amyrin synthesis hang on more than one type of BAS genes with there expression in different plant parts at different times.