Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to l-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize l-homoarginine and l-N⁶-(1-iminoethyl)lysine (l-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA^Pyl_CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host''s ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to l-homoarginine or l-NIL. The assignment of AGG to l-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.     

Identification and characterization of expressed retrotransposons in the genome of the Paracoccidioides species complex

Background

Species from the Paracoccidioides complex are thermally dimorphic fungi and the causative agents of paracoccidioidomycosis, a deep fungal infection that is the most prevalent systemic mycosis in Latin America and represents the most important cause of death in immunocompetent individuals with systemic mycosis in Brazil. We previously described the identification of eight new families of DNA transposons in Paracoccidioides genomes. In this work, we aimed to identify potentially active retrotransposons in Paracoccidioides genomes.

Results

We identified five different retrotransposon families (four LTR-like and one LINE-like element) in the genomes of three Paracoccidioides isolates. Retrotransposons were present in all of the genomes analyzed. P. brasiliensis and P. lutzii species harbored the same retrotransposon lineages but differed in their copy numbers. In the Pb01, Pb03 and Pb18 genomes, the number of LTR retrotransposons was higher than the number of LINE-like elements, and the LINE-like element RtPc5 was transcribed in Paracoccidioides lutzii (Pb01) but could not be detected in P. brasiliensis (Pb03 and Pb18) by semi-quantitative RT-PCR.

Conclusion

Five new potentially active retrotransposons have been identified in the genomic assemblies of the Paracoccidioides species complex using a combined computational and experimental approach. The distribution across the two known species, P. brasiliensis and P. lutzii, and phylogenetics analysis indicate that these elements could have been acquired before speciation occurred. The presence of active retrotransposons in the genome may have implications regarding the evolution and genetic diversification of the Paracoccidioides genus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1564-7) contains supplementary material, which is available to authorized users.     

Usabamycins A-C: new anthramycin-type analogues from a marine-derived actinomycete

New anthramycin-type analogues, designated usabamycin A-C (1, 2 and 3), have been isolated from cultures of Streptomyces sp. NPS853, a bacterium found in marine sediments. The structures of the new compounds were established on the basis of extensive spectroscopic analyses including 1D- and 2D-NMR ((1)H-(1)H COSY, HSQC, and HMBC) experiments. The usabamycins show weak inhibition of HeLa cell growth and selective inhibition of serotonin (5-hydroxytrypamine) 5-HT(2B) uptake.     

Synthesis and secretion of GnRH

Comprehensive studies have provided a clear understanding of the effects of gonadal steroids on the secretion of gonadotropin releasing hormone (GnRH), but some inconsistent results exist with regard to effects on synthesis. It is clear that regulation of both synthesis and the secretion of GnRH are effected by neurotransmitter systems in the brain. Thus, steroid regulation of GnRH synthesis and secretion can be direct, but the predominant effects are transmitted through steroid-responsive neuronal systems in various parts of the brain. There is also emerging evidence of direct effects on GnRH cells. Overriding effects on synthesis and secretion of GnRH can be observed during aging, in undernutrition and under stressful situations; these involve various neuronal systems, which may have serial or parallel effects on GnRH cells. The effect of aging is accompanied by changes in GnRH synthesis, but comprehensive studies of synthesis during undernutrition and stress are less well documented. Altered GnRH and gonadotropin secretion that occurs in seasonal breeding animals and during the pubertal transition is not generally accompanied by changes in GnRH synthesis. Secretion of GnRH from the brain is a reflection of the inherent function of GnRH cells and the inputs that integrate all of the central regulatory elements. Ultimately, the pattern of secretion dictates the reproductive status of the organism. In order to fully understand the central mechanisms that control reproduction, more extensive studies are required on the neuronal circuitry that provides input to GnRH cells.     

Homologous protein import machineries in chloroplasts and cyanelles

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria, especially in the presence of a peptidoglycan wall between the inner and outer envelope membranes. However, it is now clear that cyanelles are in fact primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario, cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high gene content of rhodoplasts and the peptidoglycan wall of cyanelles. This means that the import apparatuses of all primary plastids, i.e. those from glaucocystophytes, red algae, green algae and higher plants, should be homologous. If this is the case, then transit sequences should be similar and heterologous import experiments feasible. Thus far, heterologous in vitro import has been shown in one direction only: precursors from C. paradoxa were imported into isolated pea or spinach chloroplasts. Cyanelle transit sequences differ from chloroplast stroma targeting peptides in containing in their N-terminal domain an invariant phenylalanine residue which is shown here to be crucial for import. In addition, we now demonstrate that heterologous precursors are readily imported into isolated cyanelles, provided that the essential phenylalanine residue is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor/channel showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved, explaining the efficient heterologous import of native precursors from C. paradoxa.     

Solid-phase microextraction and chiral HPLC analysis of ibuprofen in urine

A simple and rapid solid-phase microextraction method was developed for the enantioselective analysis of ibuprofen in urine. The sampling was made with a polydimethylsiloxane-divinylbenzene coated fiber immersed in the liquid sample. After desorptioning from the fiber, ibuprofen enantiomers were analyzed by HPLC using a Chiralpak AD-RH column and UV detection. The mobile phase was made of methanol-pH 3.0 phosphoric acid solution (75:25, v/v), at a flow rate of 0.45 mL/min. The mean recoveries of SPME were 19.8 and 19.1% for (-)-R-ibuprofen and (+)-(S)-ibuprofen, respectively. The method was linear at the range of 0.25-25 microg/mL. Within-day and between-day assay precision and accuracy were below 15% for both ibuprofen enantiomers at concentrations of 0.75, 7.5 and 20 microg/mL. The method was tested with urine quality control samples and human urine fractions after administration of 200 mg rac-ibuprofen.