An engineered Tetrahymena tRNA(Gln) for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology

Ablation of PGC-1β Results in Defective Mitochondrial Activity, Thermogenesis, Hepatic Function, and Cardiac Performance

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1β (PGC-1β) has been implicated in important metabolic processes. A mouse lacking PGC-1β (PGC1βKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1βKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1β ablation was partially compensated by up-regulation of PGC-1α in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1βKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1β was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1βKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1βKO mice have impaired mitochondrial function. Lack of PGC-1β also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1β plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress

Genetic and Environmental Determinants of Wing Polymorphism in the Waterstrider \u3ci\u3eLimnoporus canaliculatus\u3c/i\u3e

Wing polymorphism commonly occurs in many insects, especially species of Orthoptera, Coleoptera, Homoptera, and Hemiptera (Harrison, 1980). The polymorphism consists of discrete differences in wing length with morphs exhibiting fully developed, reduced, or totally absent wings. In addition to differences in wing length, morphs often differ in a number of other characteristics such as degree of flight muscle development, duration of nymphal development, time to first reproduction, fertility and diapause (Anderson, 1973; Vepsäläinen, 1978; Harrison, 1980). Wing polymorphism is an attractive system for investigating the evolution of dispersal in natural populations (Vepsäläinen, 1978; Denno and Grissell, 1979; Harrison, 1980). A key step in such studies is the identification of the environmental and genetic components of morph determination. Numerous studies of insects from several different orders have clearly demonstrated that environmental variables such as photoperiod, temperature and density may strongly influence the development of an individual into a particular morph (see references in Harrison, 1980). However, the genetic component of morph determination is poorly understood. In this study we focused on the genetic influences of morph determination in the wingpolymorphic waterstrider, Limnoporus canaliculatus. We wished to determine if the inheritance of morph type could be explained by the single locus (or supergene) model proposed by most workers for other gerrid species, or whether a more complex genetic explanation was required. We used “split brood” experiments to determine if the mode of inheritance was different under different photoperiods as well as to assess the influence of photoperiod on morph determination

Evolution of a Transfer RNA Gene Through a Point Mutation in the Anticodon

The transfer RNA (tRNA) multigene family comprises 20 amino acid–accepting groups, many of which contain isoacceptors. The addition of isoacceptors to the tRNA repertoire was critical to establishing the genetic code, yet the origin of isoacceptors remains largely unexplored. A model of tRNA evolution, termed “tRNA gene recruitment,” was formulated. It proposes that a tRNA gene can be recruited from one isoaccepting group to another by a point mutation that concurrently changes tRNA amino acid identity and messenger RNA coupling capacity. A test of the model showed that an Escherichia coli strain, in which the essential tRNA_(UGU)^(Thr) gene was inactivated, was rendered viable when a tRNA^(Arg) with a point mutation that changed its anticodon from UCU to UGU (threonine) was expressed. Insertion of threonine at threonine codons by the “recruited” tRNA^(Arg) was corroborated by in vitro aminoacylation assays showing that its specificity had been changed from arginine to threonine. Therefore, the recruitment model may account for the evolution of some tRNA genes

The transfer RNA identity problem: a search for rules

Correct recognition of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases is central to the maintenance of translational fidelity. The hypothesis that synthetases recognize anticodon nucleotides was proposed in 1964 and had considerable experimental support by the mid-1970s. Nevertheless, the idea was not widely accepted until relatively recently in part because the methodologies initially available for examining tRNA recognition proved hampering for adequately testing alternative hypotheses. Implementation of new technologies has led to a reasonably complete picture of how tRNAs are recognized. The anticodon is indeed important for 17 of the 20 Escherichia coli isoaccepting groups. For many of the isoaccepting groups, the acceptor stem or position 73 (or both) is important as well

Alternative medicine and the health care division of labour: present trends and future prospects

Consumer interest in alternative medicine has expanded greatly in western societies in recent years. This has been associated, among other things, with the rapid growth of the numbers and range of practitioners of such therapies. This article discusses from a neo-Weberian perspective the present influence of this development on the nature and form of the health care division of labour, with special reference to such trends as the increasing professionalization of alternative medicine and the growing incorporation of such therapies into orthodox medical delivery systems. The article also considers the possible future impact of rising public interest in therapies currently defined as alternative on the occupational structure in health care. Of particular interest in this context is the extent to which such unorthodox practices are likely to pose a challenge to long-established patterns of biomedical dominance in the division of labour