Use Of Amino Acid Sequences Research Articles

Phylogenetic relationships among insect orders based on three nuclear protein-coding gene sequences

Many attempts to resolve the phylogenetic relationships of higher groups of insects have been made based on both morphological and molecular evidence; nonetheless, most of the interordinal relationships of insects remain unclear or are controversial. As a new approach, in this study we sequenced three nuclear genes encoding the catalytic subunit of DNA polymerase delta and the two largest subunits of RNA polymerase II from all insect orders. The predicted amino acid sequences (In total, approx. 3500 amino acid sites) of these proteins were subjected to phylogenetic analyses based on the maximum likelihood and Bayesian analysis methods with various models. The resulting trees strongly support the monophyly of Palaeoptera, Neoptera, Polyneoptera, and Holometabola, while within Polyneoptera, the groupings of Isoptera/“Blattaria”/Mantodea (Superorder Dictyoptera), Dictyoptera/Zoraptera, Dermaptera/Plecoptera, Mantophasmatodea/Grylloblattodea, and Embioptera/Phasmatodea are supported. Although Paraneoptera is not supported as a monophyletic group, the grouping of Phthiraptera/Psocoptera is robustly supported. The interordinal relationships within Holometabola are well resolved and strongly supported that the order Hymenoptera is the sister lineage to all other holometabolous insects. The other orders of Holometabola are separated into two large groups, and the interordinal relationships of each group are (((Siphonaptera, Mecoptera), Diptera), (Trichoptera, Lepidoptera)) and ((Coleoptera, Strepsiptera), (Neuroptera, Raphidioptera, Megaloptera)). The sister relationship between Strepsiptera and Diptera are significantly rejected by all the statistical tests (AU, KH and wSH), while the affinity between Hymenoptera and Mecopterida are significantly rejected only by AU and KH tests. Our results show that the use of amino acid sequences of these three nuclear genes is an effective approach for resolving the relationships of higher groups of insects.

Prediction of Number and Position of Domain Boundaries in Multi-Domain Proteins by Use of Amino Acid Sequence Alone

Prediction of protein domain boundaries is an important step for the prediction of three-dimensional structure. The simple method PDP has been elaborated for prediction of the number and position of domain boundaries in multi-domain proteins by use of amino acid sequence alone. The method uses an optimized scale based on the statistics of appearance of amino acid residues at domain boundaries. Our method demonstrates promising results in comparison to other methods that do not use homologous sequences. From the database of proteins that are targets from CASP6 (Critical Assessment of Techniques for Protein Structure Prediction) our program correctly assigned the number of domains for approximately 80% of one domain proteins and approximately 50% for two-domain proteins. Our method offers three main advantages: it is very simple, it is fast, and it uses a minimal number of parameters in comparison with other methods.

Conformational Flexibility of Pyruvate Dehydrogenase Complexes: A Computational Analysis by Quantized Elastic Deformational Model

Pyruvate dehydrogenase complex (PDC) is one of the largest multienzyme complexes known and consists of a dodecahedral E2 core to which other components are attached. We report the results of applying a new computational method, quantized elastic deformational model, to simulating the conformational fluctuations of the truncated E2 core, using low-resolution electron cryomicroscopy density maps. The motional features are well reproduced; especially, the symmetric breathing mode revealed in simulation is nearly identical with what was observed experimentally. Structural details of the motions of the trimeric building blocks, which are critical to facilitating the global expansion and contraction of the complex, were revealed. Using the low-resolution maps from electron cryomicroscopy reconstructions, the simulations showed a picture of the motional mechanism of the PDC core, which is an example without precedent of thermally activated global dynamics. Moreover, the current results support an earlier suggestion that, at low resolution and without the use of amino acid sequence and atomic coordinates, it is possible for computer simulations to provide an accurate description of protein dynamics.

Cyanobacterial sulfide-quinone reductase: cloning and heterologous expression.

The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.'), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus. SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a V(max) of 180 micromol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent K(m) values of 20 and 40 microM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding A. halophytica SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O. limnetica SQR and 437 amino acids for A. halophytica SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and -14 for O. limnetica SQR and A. halophytica SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacterium Anabaena PCC7120 as well as the chemolithotrophic bacteria Shewanella putrefaciens and Thiobacillus ferrooxidans. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter. Membranes isolated from E. coli cells expressing A. halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2n-nonyl-4-hydroxyquinoline-N-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.

Bacterial Cytochromes C and Molecular Evolution

Ambler, R. P. (Dept. of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland) 1974. Bacterial Cytochromes c and Molecular Evolution. Syst. Zool. 22: 554–565.—Cytochromes c are very widely distributed in bacteria, and many organisms produce several different types of protein. The bacterial cytochromes c vary very greatly in size, in number of hemes, in spectrum, in oxidation reduction potential, and in function, and these differences are reflected in great differences in amino acid sequence. In this paper, the present knowledge of the structure of bacterial cytochromes c is reviewed, and some new preliminary results presented. Sequence variation within classes of bacterial cytochromes c and relationships between cytochrome c classes are considered. The use of amino acid sequence results for the investigation of pre-Cambrian evolution is discussed, and pessimistic conclusions reached.