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.     

CO2 Saturation and Trophic Shift Induced by Microbial Metabolic Processes in a River-Dominated Ocean Margin (Tropical Shallow Lagoon,Chilika, India)

An effort has been made for the first time in Asia's largest brackish water lagoon, Chilika, to investigate the spatio-temporal variability in primary productivity (PP), bacterial productivity (BP), bacterial abundance (BA), bacterial respiration (BR) and bacterial growth efficiency (BGE) in relation to partial pressure of CO₂ (pCO₂) and CO₂ air–water flux and the resultant trophic switchover. Annually, PP ranged between 24 and 376 µg C L^?1 d^?1 with significantly low values throughout the monsoon (MN), caused by light limitation due to inputs of riverine suspended matter. On the contrary, BP and BR ranged from 11.5 to 186.3 µg C L^?1 d^?1 and from 14.1 to 389.4 µg C L^?1 d^?1, respectively, with exceptionally higher values during MN. A wide spatial and temporal variation in the lagoon trophic status was apparent from BP/PP (0.05–6.4) and PP/BR (0.10–18.2) ratios. The seasonal shift in net pelagic production from autotrophy to heterotrophy due to terrestrial organic matter inputs via rivers, enhanced the bacterial metabolism during the MN, as evident from the high pCO₂ (10,134 µatm) and CO₂ air–water flux (714 mm m^?2 d^?1). Large variability in BGE and BP/PP ratios especially during MN led to high bacteria-mediated carbon fluxes which was evident from significantly high bacterial carbon demand (BCD >100% of PP) during this season. This suggested that the net amount of organic carbon (either dissolved or particulate form) synthesized by primary producers in the lagoon was not sufficient to satisfy the bacterial carbon requirements. Lagoon sustained low to moderate autotrophic–heterotrophic coupling with annual mean BCD of 231% relative to the primary production, which depicted that bacterioplankton are the mainstay of the lagoon biogeochemical cycles and principal players that bring changes in trophic status. Study disclosed that the high CO₂ supersaturation and oxygen undersaturation during MN was attributed to the increased heterotrophic respiration (in excess of PP) fuelled by allochthonous organic matter. On a spatial scale, lagoon sectors such as south sector, central sector and outer channel recorded “net autotrophic,” while the northern sector showed “net heterotrophic” throughout the study period.     

Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on “active pairs” of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.     

Eighty Years of <Emphasis Type="Italic">Mycopathologia</Emphasis>: A Retrospective Analysis of Progress Made in Understanding Human and Animal Fungal Pathogens

Mycopathologia was founded in 1938 to ‘diffuse the understanding of fungal diseases in man and animals among mycologists.’ This was an important mission considering that pathogenic fungi for humans and animals represent a tiny minority of the estimated 1.5–5 million fungal inhabitants on Earth. These pathogens have diverged from the usual saprotrophic lifestyles of most fungi to colonize and infect humans and animals. Medical and veterinary mycology is the subdiscipline of microbiology that dwells into the mysteries of parasitic, fungal lifestyles. Among the oldest continuing scientific publications on the subject, Mycopathologia had its share of ‘classic papers’ since the first issue was published in 1938. An analysis of the eight decades of notable contributions reveals many facets of host–pathogen interactions among 183 volumes comprising about 6885 articles. We have analyzed the impact and relevance of this body of work using a combination of citation tools (Google Scholar and Scopus) since no single citation metric gives an inclusive perspective. Among the highly cited Mycopathologia publications, those on experimental mycology accounted for the major part of the articles (36%), followed by diagnostic mycology (16%), ecology and epidemiology (15%), clinical mycology (14%), taxonomy and classification (10%), and veterinary mycology (9%). The first classic publication, collecting nearly 200 citations, appeared in 1957, while two articles published in 2010 received nearly 150 citations each, which is notable for a journal covering a highly specialized field of study. An empirical analysis of the publication trends suggests continuing interests in novel diagnostics, fungal pathogenesis, review of clinical diseases especially with relevance to the laboratory scientists, taxonomy and classification of fungal pathogens, fungal infections and carriage in pets and wildlife, and changing ecology and epidemiology of fungal diseases around the globe. We anticipate that emerging and re-emerging fungal pathogens will continue to cause significant health burden in the coming decades. It remains vital that scientists and physicians continue to collaborate by learning each other’s language for the study of fungal diseases, and Mycopathologia will strive to be their partner in this increasingly important endeavor to its 100th anniversary in 2038 and beyond.     

Simple and efficient genomic DNA extraction protocol for molecular characterisation of Phytophthora colocasiae causing taro leaf blight

Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A_260/280 and A_260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers.     

Ubiquitylation Pathways In Insulin Signaling and Organismal Homeostasis

The insulin/insulin‐like growth factor‐1 (IGF‐1) signaling (IIS) pathway is a pivotal genetic program regulating cell growth, tissue development, metabolic physiology, and longevity of multicellular organisms. IIS integrates a fine‐tuned cascade of signaling events induced by insulin/IGF‐1, which is precisely controlled by post‐translational modifications. The ubiquitin/proteasome‐system (UPS) influences the functionality of IIS through inducible ubiquitylation pathways that regulate internalization of the insulin/IGF‐1 receptor, the stability of downstream insulin/IGF‐1 signaling targets, and activity of nuclear receptors for control of gene expression. An age‐related decline in UPS activity is often associated with an impairment of IIS, contributing to pathologies such as cancer, diabetes, cardiovascular, and neurodegenerative disorders. Recent findings identified a key role of diverse ubiquitin modifications in insulin signaling decisions, which governs dynamic adaption upon environmental and physiological changes. In this review, we discuss the mutual crosstalk between ubiquitin and insulin signaling pathways in the context of cellular and organismal homeostasis.     

Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: Role of dark respiration and of light

Effect of quality, quantity and minimum duration of light on the process of recovery was investigated in the photoinhibited cells of the green alga Chlamydomonas reinhardtii. Complete and rapid reactivation of photosynthesis took place in diffuse white light of 25 mol m^–2 s^–1. The recovery was partial (< 10%) in the dark. Far red (725 nm), red (660 nm) and blue light (480 nm) in the range of 10 to 75 mol m^–2 s^–1 did not enhance the process of reactivation. Photoinhibited cells incubated in dark for 15 min when exposed for 5 min to diffuse light (25 mol m^–2 s^–1) showed complete reactivation. Even exposure of 15 min dark incubated photoinhibited cells to photoinhibitory light (2500 mol m^–2 s^–1) for 5 s fully regained the photosynthesis. The study indicated a very precise and triggering effect of light in the process of reactivation. The dark respiratory inhibitor KCN and uncouplers FCCP and CCCP increased the susceptibility of C. reinhardtii to photoinhibition and also prevented photoinhibited cells to reactivate fully even after longer period of incubation under suitable reactivating conditions. Of the various possibilities envisaged to assign the role of dark respiration in recovery process, supply of ATP by mitochondrial respiration appeared sound and pertinent.Abbreviations CCCP- carbonyl cyanide m-chlorophenylhydrazone - D1- 32 kDa protein of PS II reaction center - FCCP- carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone - KCN- potassium cyanide - PBQ- phenyl-p-benzoquinone - PFD- photon flux density - SHAM- salicylhydroxamic acid NBRI Research Publication No. 431.