Engineering an Isoprenoid Pathway in Escherichia coli for Production of 2-Methyl-3-buten-2-ol: A Potential Biofuel

2-Methyl-3-buten-2-ol (MBO) is a natural volatile 5-carbon alcohol produced by several pine species that have the potential to be used as biofuel. MBO has a high energy content making it superior to ethanol in terms of energy output, and due to its volatility and lower solubility in water, MBO is easier to recover than ethanol. Pine’s MBO synthase enzyme utilizes the intermediate dimethylallyl pyrophosphate (DMAPP) produced by the methyl-erythritol-4-phosphate isoprenoid pathway for the production of MBO. In this study, we performed metabolic engineering of Escherichia coli to express an alternate mevalonate dependent pathway for production of DMAPP, along with a codon optimized Pinus sabiniana MBO synthase gene. This heterologous expressed pathway carried out the conversion of an acetyl CoA precursor to DMAPP leading to production of MBO.     

Protein–protein association rates captured in a single geometric parameter

Understanding factors that drive protein–protein association is of fundamental importance. We show that a single geometric parameter in crystal structures of protein–protein complexes, the angle between the electric dipole of one subunit and the partner‐generated electric field at the same subunit, linearly correlates with experimentally determined protein–protein association rates. Imprint of a dynamic kinetic process in a single static geometric parameter, associated with mutual electrostatic orientation of subunits in protein–protein complexes, is elegant and demonstrates the universality of electrostatic steering in attenuating protein–protein association rates. That the essence of a complex phenomenon could be captured by properties of the final crystal structure of the complex implies that the electrostatic orientations of protein subunits in crystal structures and the associated transition states are nearly identical. Further, the cosine of the angle, alone, is shown to be sufficient in predicting association rate constants, with accuracies comparable to currently available predictors that use more intricate methodologies. Our results offer mechanistic insights and could be useful in development of coarse‐grained models. Proteins 2015; 83:1557–1562. © 2015 Wiley Periodicals, Inc.     

Requirement of rRNA methylation for 80S ribosome assembly on a cohort of cellular internal ribosome entry sites

Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.     

MAPS: an interactive web server for membrane annotation of transmembrane protein structures

The exact positioning of the membrane in transmembrane (TM) proteins plays important functional roles. Yet, the structures of TM proteins in protein data bank (pdb) have no information about the explicit position of the membrane. Using a simple hydrophobic lipid-protein mismatch energy function and a flexible lipid/water boundary, the position of lipid bilayer for representative TM proteins in pdb have been annotated. A web server called MAPS (Membrane Annotation of Protein Structures; available at: http://www.boseinst.ernet.in/gautam/maps) has been set up that allows the user to interactively analyze membrane-protein orientations of any uploaded pdb structure with user-defined membrane flexibility parameters.     

Studies on the interaction of the food colorant tartrazine with double stranded deoxyribonucleic acid

Interaction of the food additive tartrazine with double-stranded DNA was studied by spectroscopic and calorimetric techniques. Absorbance studies revealed that tartrazine exhibited hypochromism in the presence of DNA without any bathochromic effects. Minor groove displacement assay of DAPI and Hoechst 33258 suggested that tartrazine binds in the minor groove of DNA. The complexation was predominantly entropy driven with a smaller but favorable enthalpic contribution to the standard molar Gibbs energy. The equilibrium constant was evaluated to be (3.68?±?.08)?×?10⁴?M^?1 at 298.15?K. The negative standard molar heat capacity value along with an enthalpy–entropy compensation phenomenon proposed the involvement of dominant hydrophobic forces in the binding process. Tartrazine enhanced the thermal stability of DNA by 7.53?K under saturation conditions.     

The Des-1 protein, required for central spindle assembly and cytokinesis, is associated with mitochondria along the meiotic spindle apparatus and with the contractile ring during male meiosis in Drosophila melanogaster

Spermatogenesis in Drosophila melanogaster serves as an excellent model system for the isolation and analysis of genes required in the control of chromosome segregation and cytokinesis. We report here the isolation and molecular characterization of a novel P-element induced allele of the des-1 gene, which leads to male sterility as a consequence of the failure of central spindle assembly in meiotic spermatocytes and the formation of aberrant meiotic end products characteristic of cytokinesis failure. We have raised affinity-purified antibodies against a Des-1 fusion protein, and localized the Des-1 protein in Drosophila spermatocytes. We show that the Des-1 protein is colocalized with mitochondria throughout male meiosis, becoming intimately associated with mitochondria along the spindle apparatus during anaphase and telophase, and with the Nebenkern, or mitochondrial derivative, of the meiotic end products. In addition, a significant association of Des-1 with the contractile ring is observed during anaphase and telophase of meiosis. These observations, together with the presence of six potential transmembrane domains in the Des-1 protein, raise the possibility that Des-1 may act as part of an anchoring mechanism that links membrane-bounded cellular compartments to components of the cytoskeleton.     

Bucking the trend: the African Black Oystercatcher as a recent conservation success story

The African Black Oystercatcher Haematopus moquini is a charismatic, southern African near-endemic, wader species, that is often seen as a flagship species for coastal bird conservation, as it was recently down-listed regionally to Least Concern on the IUCN Red List of Threatened Species. To celebrate this rare conservation success story, BirdLife South Africa named it the 2018 Bird of the Year and ran a year-long programme in collaboration with the Nature’s Valley Trust highlighting aspects of the species’ biology, current threats, and conservation success. We used data collected by the Southern African Bird Atlas Project (SABAP1 and SABAP2) to examine changes in the species’ range and relative abundance, both in the records between the two projects, as well as trends within the SABAP2 sampling period (2008–2017). This case study enabled us to assess whether such metrics can accurately reflect abundance and distributional changes in a species. We found increases in the reported range and the reporting rates between the two Atlas projects, and that the SABAP2 reporting rate was stable. Regionally, across four coastal categories, the reporting rate was lowest in KwaZulu-Natal, though this region also showed an increase in the probability of reporting during the SABAP2 period. While corroborating the recent change in the species’ conservation status, we also provide good evidence that the long-term SABAP data can be used successfully to assess population trends and range changes over time.     

Identification of QTLs for Yield and Associated Traits in F₂ Population of Rice

Identification of quantitative trait loci (QTLs) controlling yield and yield-related traits in rice was performed in the F₂ mapping population derived from parental rice genotypes DHMAS and K343. A total of 30 QTLs governing nine different traits were identified using the composite interval mapping (CIM) method. Four QTLs were mapped for number of tillers per plant on chromosomes 1 (2 QTLs), 2 and 3; three QTLs for panicle number per plant on chromosomes 1 (2 QTLs) and 3; four QTLs for plant height on chromosomes 2, 4, 5 and 6; one QTL for spikelet density on chromosome 5; four QTLs for spikelet fertility percentage (SFP) on chromosomes 2, 3 and 5 (2 QTLs); two QTLs for grain length on chromosomes 1 and 8; three QTLs for grain width on chromosomes1, 3 and 8; three QTLs for 1000-grain weight (TGW) on chromosomes 1, 4 and 8 and six QTLs for yield per plant (YPP) on chromosomes 2 (3 QTLs), 4, 6 and 8. Most of the QTLs were detected on chromosome 2, so further studies on chromosome 2 could help unlock some new chapters of QTL for this cross of rice variety. Identified QTLs elucidating high phenotypic variance can be used for marker-assisted selection (MAS) breeding. Further, the exploitation of information regarding molecular markers tightly linked to QTLs governing these traits will facilitate future crop improvement strategies in rice.     

Heregulin-induced epigenetic regulation of the utrophin-A promoter

Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over-expression can compensate dystrophin's absence. Utrophin is enriched at neuromuscular junctions due to heregulin-mediated utrophin-A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK-dependent manner. MSK1/2 inhibition suppressed heregulin-mediated utrophin-A activation. MSK1 over-expression potentiated heregulin-mediated utrophin-A activation and chromatin remodeling at the utrophin-A promoter. These results identify MSK1/2 as key effectors modulating utrophin-A expression as well as identify novel targets for DMD therapy.