Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Trichloroacetaldehyde modified oligonucleotides

Some commercial batches of dichloroacetic acid (DCA) contain traces of chloral (trichloroacetaldehyde). Using such DCA to effect detritylation during solid-phase oligonucleotide synthesis results in the formation of a family of process impurities in which the atoms of chloral (Cl3CCHO) are incorporated between the 5'-oxygen and phosphorus atoms of an internucleotide linkage. The structure was elucidated by HPLC with UV and MS detection, digestion of the oligonucleotide, synthesis of model compounds, and 1H and 31P NMR spectroscopy. By understanding the chemistry behind its formation, we are now able to limit levels of this impurity in synthetic oligonucleotides by limiting chloral in DCA.     

Caudal autotomy and regeneration in lizards

Caudal autotomy, or the voluntary self-amputation of the tail, is an anti-predation strategy in lizards that depends on a complex array of environmental, individual, and species-specific characteristics. These factors affect both when and how often caudal autotomy is employed, as well as its overall rate of success. The potential costs of autotomy must be weighed against the benefits of this strategy. Many species have evolved specialized behavioral and physiological adaptations to minimize or compensate for any negative consequences. One of the most important steps following a successful autotomous escape involves regeneration of the lost limb. In some species, regeneration occurs rapidly; such swift regeneration illustrates the importance of an intact, functional tail in everyday experience. In lizards and other vertebrates, regeneration is a highly ordered process utilizing initial developmental programs as well as regeneration-specific mechanisms to produce the correct types and pattern of cells required to sufficiently restore the structure and function of the sacrificed tail. In this review, we discuss the behavioral and physiological features of self-amputation, with particular reference to the costs and benefits of autotomy and the basic mechanisms of regeneration. In the process, we identify how these behaviors could be used to explore the neural regulation of complex behavioral responses within a functional context.     

TOR-tured Yeast Find a New Way to Stand the Heat

In this issue, Takahara and Maeda (2012) discover that together, Pbp1 and sequestration of the TORC1 complex in cytoplasmic mRNP stress granules provides a negative regulatory mechanism for TORC1 signaling during stress.     

An immunocytochemical approach to detection of mitochondrial disorders.

Mitochondrial disorders can lead to a confusing array of symptoms, which frequently makes a diagnosis difficult. Traditional approaches to such diagnoses are based on enzyme activity assays, with further characterization provided by genetic analysis. However, these methods require relatively large sample sizes, are time-consuming, labor-intensive, and show variability between laboratories. Here, we report an immunocytochemical test that makes use of monoclonal antibodies to subunits from each of the oxidative phosphorylation complexes and pyruvate dehydrogenase to aid in the detection of mitochondrial disorders. It can be completed and data analyzed in less than 4 hr. We have used this test to study fibroblast cultures from patients with mitochondrial disorders arising from both mitochondrial DNA and nuclear DNA defects. We have also examined cases of Leigh syndrome arising from different genetic causes. We show that patients can be categorized on the basis of which complexes are affected and whether or not the defect being studied shows a mosaic distribution, an indicator of whether the causal mutation(s) is/are in the mitochondrial or nuclear genome. Immunocytochemical analysis as described here should be considered as an initial screen for mitochondrial disorders by which to direct (and limit) the subsequent enzymatic and genetic tests required to make an unambiguous diagnosis.     

Three-dimensional structures of cytochrome c oxidase vesicle crystals in negative stain

We have investigated the structure of cytochrome c oxidase vesicle crystals by analysis at 20 Å resolution of electron micrographs of negatively stained specimens. The map clearly shows the shape of the part of the cytochrome c oxidase molecule which protrudes from the lipid bilayer. On the side of the membrane corresponding to the cytoplasmic face of the mitochondrial inner membrane, the molecule projects over 50 Å into solution. About half of the mass of the protein is in this domain, which contains the cytochrome c binding site. On the side of the membrane corresponding to the matrix face, no features are observed, which at this resolution means the protein protrudes less than 20 Å. In vesicle crystals, and probably in the mitochondrion, cytochrome c oxidase monomers are closely paired as dimers, with a clear cleft showing the boundary between monomers.     

Mechanism of the F(1)F(0)-type ATP synthase, a biological rotary motor

The F(1)F(0)-type ATP synthase is a key enzyme in cellular energy interconversion. During ATP synthesis, this large protein complex uses a proton gradient and the associated membrane potential to synthesize ATP. It can also reverse and hydrolyze ATP to generate a proton gradient. The structure of this enzyme in different functional forms is now being rapidly elucidated. The emerging consensus is that the enzyme is constructed as two rotary motors, one in the F(1) part that links catalytic site events with movements of an internal rotor, and the other in the F(0) part, linking proton translocation to movements of this F(0) rotor. Although both motors can work separately, they must be connected together to interconvert energy. Evidence for the function of the rotary motor, from structural, genetic and biophysical studies, is reviewed here, and some uncertainties and remaining mysteries of the enzyme mechanism are also discussed.