Sandhya Mehrotra

Designer promoter: an artwork of cis engineering

Advances in systematic computational biology and rapid elucidation of synergistic interplay between cis and trans factors governing transcriptional control have facilitated functional annotation of gene networks. The generation of data through deconstructive, reconstructive and database assisted promoter studies, and its integration to principles of synthetic engineering has started an era of designer promoters. Exploration of natural promoter architecture and the concept of cis engineering have not only enabled fine tuning of single or multiple transgene expression in response to perturbations in the chemical, physiological and environmental stimuli but also provided researchers with a unique answer to various problems in crop improvement in the form of bidirectional promoters.

Promoter activation by ACGT in response to salicylic and abscisic acids is differentially regulated by the spacing between two copies of the motif

A variety of small sequence motifs, located upstream of minimal promoters influence gene expression. The function of the core sequence ACGT, present in a family of commonly occurring cis acting transcription regulatory motifs was investigated in the background of an artificially designed synthetic promoter sequence. The ACGT was placed in one or two copies and separated by different spacer lengths between the two copies, to study their affect on the expression of basal promoter in plant cells. The activation of transcription by the ACGT element was examined by transient and stable transformation in tobacco, using gusA as the reporter gene. The analysis shows that the expression of the reporter gene was influenced differently by spacing between two adjacent copies of the motif. Two copies of the ACGT element separated by 5 nucleotides gave highest activation. This configuration imparted salicylic acid inducibility to the basal promoter. However, two copies of ACGT separated by 25 nucleotides allowed the promoter to be induced by abscisic acid but not salicylic acid. Computational analysis of the Arabidopsis thaliana genome database showed the presence of these motifs in several genes associated with a variety of stress responses. The results on motif-related inducibility by salicylic acid and abscisic acid, as seen in a synthetic sequence background were validated by experiments on the expression of the native promoter of a protein phosphatase 2C-like gene of Arabidopsis in tobacco leaves. The study supports the importance of spacing between the ACGT sequence motifs on the elicitor specific modulation of gene expression and demonstrates the role of different ACGT motifs in the regulation of PP2C gene expression.

Developments and challenges in biodiesel production from microalgae: A review

The imminent depletion of fossil fuels and the surging global demand for renewable energy have led to the search for nonconventional energy sources. After a few decades of trial and error, the world is now testing the sources of the third generation of fossil fuels, which contain for most parts microalgae. With more than 80% oil content, being adaptable in growth parameters and highly versatile, microalgae are highly promising sources of biofuels in the present time. The present article makes a sweeping attempt to highlight the various methods employed for cultivation of microalgae, techniques to harvest and extract biomass from huge algal cultures, as well as their downstream production and processing procedures. The advantages, limitations, and challenges faced by each of them have been described to some extent. Major concerns pertaining to biofuels are supposed to be their environmental sustainability and economic viability along with their cost effectiveness. This would require a great deal of empirical data on existing systems and a great deal of optimization to generate a more robust one. We have concluded our article with a SWOT analysis of using algae for biodiesel production in a tabulated form.

Lipase Immobilization Techniques for Biodiesel Production: An Overview

The growing energy needs and depleting fuel sources compel us to look towards production of biodiesel, an appropriate alternative. The industrially used chemical catalysis process is beset with problems that enzymatic production using lipases could avoid. In this light, the immobilization of lipases plays an important role in the optimization of the production process. This review discusses the various techniques that have been studied for lipase immobilization, namely adsorption, covalent attachment, entrapment, cross-linked enzyme agglomerates and whole-cell biocatalysts, while highlighting their benefits and drawbacks. It also sheds light on the future of enzyme immobilization and its industrial application.

Patterns and evolution of ACGT repeat cis-element landscape across four plant genomes

BackgroundTranscription factor binding is regulated by several interactions, primarily involving cis-element binding. These binding sites maintain specificity by means of their sequence, and other additional factors such as inter-motif distance and spacer specificity. The ACGT core sequence has been established as a functionally important cis-element which frequently regulates gene expression in synergy with other cis-elements. In this study, we used two monocotyledonous – Oryza sativa and Sorghum bicolor, and two dicotyledonous species – Arabidopsis thaliana and Glycine max to analyze the conservation of co-occurring ACGT core elements in plant promoters with respect to spacer distance between them. Using data generated from Arabidopsis thaliana and Oryza sativa, we also identified conserved regions across all spacers and possible conditions regulating gene promoters with multiple ACGT cis-elements.ResultsOur data indicated specific predominant spacer lengths between co-occurring ACGT elements, but these lengths were not universally conserved across all species under analysis. However, the frequency distribution indicated local regions of high correlation among monocots and dicots. Sequence specificity data clearly revealed a preference for G at the first and C at the terminal position of a spacer sequence, suggesting that the G-box motif is the most prevalent for the ACGT class of promoters. Using gene expression databases, we also observed trends suggesting that co-occurring ACGT elements are responsible for gene regulation in response to exogenous stress. Conservation in patterns of ACGT (N) ACGT among orthologous genes also indicated the possibility that emergence of functional significance across species was a result of parallel evolution of these cis-elements.ConclusionsAlthough the importance of ACGT elements has been acknowledged for several plant species, ours is the first study that attempts to compare their occurrence across four species and analyze conservation among them. The apparent preference for particular spacer distances suggest that these motifs might be implicated in important physiological functions which are yet to be identified. Variations in correlation patterns among monocots and dicots might arise out of differences in transcriptional regulation in the two classes. In accordance with literature, we established the involvement of co-occurring ACGT elements in stress responses and showed how this regulation differs with variation in the ACGT (N) ACGT motif. We believe that our study will be an essential resource in determining optimum spacer length and spacer sequence between ACGT elements for promoter design in future.

Combinatorial Control of Gene Expression

The complexity and diversity of eukaryotic organisms are a feat of natures engineering. Pulling the strings of such an intricate machinery requires an even more masterful and crafty approach. Only the number and type of responses that they generate exceed the staggering proportions of environmental signals perceived and processed by eukaryotes. Hence, at first glance, the cells sparse stockpile of controlling factors does not seem remotely adequate to carry out this response. The question as to how eukaryotes sense and respond to environmental cues has no single answer. It is an amalgamation, an interplay between several processes, pathways, and factors—a combinatorial control. A short description of some of the most important elements that operate this entire conglomerate is given in this paper.

Evidence for Directed Evolution of Larger Size Motif in Arabidopsis thaliana Genome

Transcription control of gene expression depends on a variety of interactions mediated by the core promoter region, sequence specific DNA-binding proteins, and their cognate promoter elements. The prominent group of cis acting elements in plants contains an ACGT core. The cis element with this core has been shown to be involved in abscisic acid, salicylic acid, and light response. In this study, genome-wide comparison of the frequency of occurrence of two ACGT elements without any spacers as well as those separated by spacers of different length was carried out. In the first step, the frequency of occurrence of the cis element sequences across the whole genome was determined by using BLAST tool. In another approach the spacer sequence was randomized before making the query. As expected, the sequence ACGTACGT had maximum occurrence in Arabidopsis thaliana genome. As we increased the spacer length, one nucleotide at a time, the probability of its occurrence in genome decreased. This trend continued until an unexpectedly sharp rise in frequency of (ACGT)N25(ACGT). The observation of higher probability of bigger size motif suggests its directed evolution in Arabidopsis thaliana genome.

Towards combinatorial transcriptional engineering

The modular nature of the transcriptional unit makes it possible to design robust modules with predictable input-output characteristics using a ‘parts- off a shelf’ approach. Customized regulatory circuits composed of multiple such transcriptional units have immense scope for application in diverse fields of basic and applied research. Synthetic transcriptional engineering seeks to construct such genetic cascades. Here, we discuss the three principle strands of transcriptional engineering: promoter and transcriptional factor engineering, and programming inducibilty into synthetic modules. In this context, we review the scope and limitations of some recent technologies that seek to achieve these ends. Our discussion emphasizes a requirement for rational combinatorial engineering principles and the promise this approach holds for the future development of this field.

Genome wide analysis of Arabidopsis thaliana reveals high frequency of AAAGN7CTTT motif

Sequence specific elements in DNA regulate transcription by recruiting transcription factors. The Dof proteins are a large family of transcription factors that share a single highly conserved zinc finger. The core to which Dof proteins bind has a consensus AAAG or ACTTTA sequence. These motifs have been over represented in many promoters. We performed a genome wide analysis of AAAG repeat elements increasing the spacer length from 0 to 25. Similar analyses was done with AAAG-CTTT motifs. We report unusual high frequency of AAAGN7CTTT in Arabidopsis thaliana genome. We also conclude that there is a preference for A/G nucleotides in spacer sequence between two AAAG repeats.

Regulatory components of carbon concentrating mechanisms in aquatic unicellular photosynthetic organisms

This review provides an insight into the regulation of the carbon concentrating mechanisms (CCMs) in lower organisms like cyanobacteria, proteobacteria, and algae. CCMs evolved as a mechanism to concentrate CO2 at the site of primary carboxylating enzyme Ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco), so that the enzyme could overcome its affinity towards O2 which leads to wasteful processes like photorespiration. A diverse set of CCMs exist in nature, i.e., carboxysomes in cyanobacteria and proteobacteria; pyrenoids in algae and diatoms, the C4 system, and Crassulacean acid metabolism in higher plants. Prime regulators of CCM in most of the photosynthetic autotrophs belong to the LysR family of transcriptional regulators, which regulate the activity of the components of CCM depending upon the ambient CO2 concentrations. Major targets of these regulators are carbonic anhydrase and inorganic carbon uptake systems (CO2 and HCO3− transporters) whose activities are modulated either at transcriptional level or by changes in the levels of their co-regulatory metabolites. The article provides information on the localization of the CCM components as well as their function and participation in the development of an efficient CCM. Signal transduction cascades leading to activation/inactivation of inducible CCM components on perception of low/high CO2 stimuli have also been brought into picture. A detailed study of the regulatory components can aid in identifying the unraveled aspects of these mechanisms and hence provide information on key molecules that need to be explored to further provide a clear understanding of the mechanism under study.