High-level expression of murine terminal deoxynucleotidyl transferase inEscherichia coli grown at low temperature and overexpressingargU tRNA

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5′-triphosphates onto the 3′-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production inEscherichia coli of murine TdT with minimal proteolysis and high specific activity.     

Nature of selective constraints on synonymous codon usage of rice differs in GC-poor and GC-rich genes

Synonymous codon usage and cellular tRNA abundance are thought to be co-evolved in optimizing translational efficiencies in highly expressed genes. Here in this communication by taking the advantage of publicly available gene expression data of rice and Arabidopsis we demonstrated that tRNA gene copy number is not the only driving force favoring translational selection in all highly expressed genes of rice. We found that forces favoring translational selection differ between GC-rich and GC-poor classes of genes. Supporting our results we also showed that, in highly expressed genes of GC-poor class there is a perfect correspondence between majority of preferred codons and tRNA gene copy number that confers translational efficiencies to this group of genes. However, tRNA gene copy number is not fully consistent with models of translational selection in GC-rich group of genes, where constraints on mRNA secondary structure play a role to optimize codon usage in highly expressed genes.     

Targeted expression of redesigned and codon optimised synthetic gene leads to recrystallisation inhibition and reduced electrolyte leakage in spring wheat at sub-zero temperatures

Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 α-helical antifreeze protein from winter flounder was rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06–0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targeted antifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as −7°C.     

Roost tree characteristics determine use by the white‐striped freetail bat (Tadarida australis,Chiroptera: Molossidae) in suburban subtropical Brisbane,Australia

Abstract We examined factors affecting roost tree selection by the white‐striped freetail bat Tadarida australis (Chiroptera: Molossidae), a large insectivorous bat in suburban Brisbane, Australia. We compared biophysical characteristics associated with 34 roost trees and 170 control trees of similar diameter, height and tree senescence characters. Roost trees used by the white‐striped freetail bat had significantly higher numbers of hollows in the trunk and branches (P < 0.003) and were more likely to contain a large trunk cavity with an internal diameter of >30 cm (P < 0.001) than control trees. These trees also accommodated more species of hollow‐using fauna (P = 0.005). When comparing roost trees with control trees of similar diameters and heights, roost trees were on average at a later stage of tree senescence (P < 0.001). None of the roost trees were found in the large forest reserves fringing the Brisbane metropolitan area despite these areas being used for foraging by the white‐striped freetail bat. Although all tree locations in this study were in modified landscapes, roost trees tended to be surrounded by groups of trees and undergrowth. Roost trees provide important habitat requirements for hollow‐using fauna in suburban, rural and forested environments.     

Lactic acid bacteria as prime candidates for codon optimization

In species having a strong correlation of expressivity and codon bias it has been shown that heterologous expression can be optimized by changing codons of the introduced gene towards the set of codons that the host organism naturally uses in its highly expressed genes. Even though two lactic acid bacteria are fully sequenced, there are no reports on attempts of codon optimization in the literature. In this report it is demonstrated that codons used in highly expressed genes tend to differ from the codons in lowly expressed genes, and that there is a strong correlation of codon bias and empirical expressivity (codon adaptation index) in Lactococcus lactis and Lactobacillus plantarum. This strongly suggests that codon optimization strategies could be applied to expression systems with lactic acid bacteria as producer strains. A good example of a candidate for codon optimization is the mouse interleukin-2 gene, which in its natural form has an extremely low codon adaptation index for expression in Lc. lactis.     

Molecular characterization influencing metal resistance in the Cupriavidus/Ralstonia genomes

Our environment is stressed with a load of heavy and toxic metals. Microbes, abundant in our environment, are found to adapt well to this metal-stressed condition. A comparative study among five Cupriavidus/Ralstonia genomes can offer a better perception of their evolutionary mechanisms to adapt to these conditions. We have studied codon usage among 1051 genes common to all these organisms and identified 15 optimal codons frequently used in highly expressed genes present within 1051 genes. We found the core genes of Cupriavidus metallidurans CH34 have a different optimal codon choice for arginine, glycine and alanine in comparison with the other four bacteria. We also found that the synonymous codon usage bias within these 1051 core genes is highly correlated with their gene expression. This supports that translational selection drives synonymous codon usage in the core genes of these genomes. Synonymous codon usage is highly conserved in the core genes of these five genomes. The only exception among them is C. metallidurans CH34. This genomewide shift in synonymous codon choice in C. metallidurans CH34 may have taken place due to the insertion of new genes in its genomes facilitating them to survive in heavy metal containing environment and the co-evolution of the other genes in its genome to achieve a balance in gene expression. Structural studies indicated the presence of a longer N-terminal region containing a copper-binding domain in the cupC proteins of C. metallidurans CH3 that helps it to attain higher binding efficacy with copper in comparison with its orthologs.     

Expression in Escherichia coli of Biphenyl 2,3-Dioxygenase Genes from a Gram-Positive Polychlorinated Biphenyl Degrader,Rhodococcus jostii RHA1

Rhodococcus jostii RHA1 is a polychlorinated biphenyl degrader. Multi-component biphenyl 2,3-dioxygenase (BphA) genes of RHA1 encode large and small subunits of oxygenase component and ferredoxin and reductase components. They did not express enzyme activity in Escherichia coli. To obtain BphA activity in E. coli, hybrid BphA gene derivatives were constructed by replacing ferredoxin and/or reductase component genes of RHA1 with those of Pseudomonas pseudoalcaligenes KF707. The results obtained indicate a lack of catalytic activity of the RHA1 ferredoxin component gene, bphAc in E. coli. To determine the cause of inability of RHA1 bphAc to express in E. coli, the bphAc gene was introduced into Rosetta (DE3) pLacI, which has extra tRNA genes for rare codons in E. coli. The resulting strain abundantly produced the bphAc product, and showed activity. These results suggest that codon usage bias is involved in inability of RHA1 bphAc to express its catalytic activity in E. coli.     

Nucleosomal Positioning and Genetic Divergence Study Based on DNA Flexibility Map

Abstract

Based on worm like chain model, DNA structural parameters—tilt, roll and rise, derived from crystallographic database have been used to determine the flexibility of DNA that regulates the nucleosomal translational positioning. Theoretically derived data has been compared to the experimental values available in Ioshikhes and Trifonov's database. The methodology has been extended to determine the flexibility of 18S rRNA genome in eukarya, where yeast shows a distinct difference when compared with mammals like human, mouse and rabbit.