Shigeru Muta

Evolutionary changes of multiple visual pigment genes in the complete genome of Pacific bluefin tuna.

Tunas are migratory fishes in offshore habitats and top predators with unique features. Despite their ecological importance and high market values, the open-ocean lifestyle of tuna, in which effective sensing systems such as color vision are required for capture of prey, has been poorly understood. To elucidate the genetic and evolutionary basis of optic adaptation of tuna, we determined the genome sequence of the Pacific bluefin tuna (Thunnus orientalis), using next-generation sequencing technology. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified five common fish visual pigment genes: red-sensitive (middle/long-wavelength sensitive; M/LWS), UV-sensitive (short-wavelength sensitive 1; SWS1), blue-sensitive (SWS2), rhodopsin (RH1), and green-sensitive (RH2) opsin genes. Sequence comparison revealed that tunas RH1 gene has an amino acid substitution that causes a short-wave shift in the absorption spectrum (i.e., blue shift). Pacific bluefin tuna has at least five RH2 paralogs, the most among studied fishes; four of the proteins encoded may be tuned to blue light at the amino acid level. Moreover, phylogenetic analysis suggested that gene conversions have occurred in each of the SWS2 and RH2 loci in a short period. Thus, Pacific bluefin tuna has undergone evolutionary changes in three genes (RH1, RH2, and SWS2), which may have contributed to detecting blue-green contrast and measuring the distance to prey in the blue-pelagic ocean. These findings provide basic information on behavioral traits of predatory fish and, thereby, could help to improve the technology to culture such fish in captivity for resource management.

The hydrophobic cores of proteins predicted by wavelet analysis.

MOTIVATION In the process of protein construction, buried hydrophobic residues tend to assemble in a core of a protein. Methods used to predict these cores involve use or no use of sequential alignment. In the case of a close homology, prediction was more accurate if sequential alignment was used. If the homology was weak, predictions would be unreliable. A hydrophobicity plot involving the hydropathy index is useful for purposes of prediction, and smoothing is essential. However, the proposed methods are insufficient. We attempted to predict hydrophobic cores with a low frequency extracted from the hydrophobicity plot, using wavelet analysis. RESULTS The cores were predicted at a rate of 68.7%, by cross-validation. Using wavelet analysis, the cores of non-homologous proteins can be predicted with close to 70% accuracy, without sequential alignment. AVAILABILITY The program used in this study is available from Intergalactic Reality (http://www.intergalact.com). CONTACT hirakawa@grt.kyushu-u.ac.jp, kuhara@grt.kyushu-u.ac.jp

Changes at the floor of the peptide-binding groove induce a strong preference for proline at position 3 of the bound peptide: molecular dynamics simulations of HLA-A*0217.

We report on molecular dynamics simulations of major histocompatibility complex (MHC)-peptide complexes. Class I MHC molecules play an important role in cellular immunity by presenting antigenic peptides to cytotoxic T cells. Pockets in the peptide-binding groove of MHC molecules accommodate anchor side chains of the bound peptide. Amino acid substitutions in MHC affect differences in the peptide-anchor motifs. HLA-A*0217, human MHC class I molecule, differs from HLA-A*0201 only by three amino acid residues substitutions (positions 95, 97, and 99) at the floor of the peptide-binding groove. A*0217 showed a strong preference for Pro at position 3 (p3) and accepted Phe at p9 of its peptide ligands, but these preferences have not been found in other HLA-A2 ligands. To reveal the structural mechanism of these observations, the A*0217-peptide complexes were simulated by 1000 ps molecular dynamics at 300 K with explicit solvent molecules and compared with those of the A*0201-peptide complexes. We examined the distances between the anchor side chain of the bound peptide and the pocket, and the rms fluctuations of the bound peptides and the HLA molecules. On the basis of the results from our simulations, we propose that Pro at p3 serves as an optimum residue to lock the dominant anchor residue (p9) tightly into pocket F and to hold the peptide in the binding groove, rather than a secondary anchor residue fitting optimally the complementary pocket. We also found that Phe at p9 is used to occupy the space created by replacements of three amino acid residues at the floor within the groove. These findings would provide a novel understanding in the peptide-binding motifs of class I MHC molecules.

Properties of a Trifluoroleucine-Resistant Mutant of Saccharomyces cerevisiae

We characterized a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae, TFL20, that has a mutation in the LEU4 gene. We monitored the concentration of extracellular i-AmOH and intracellular amino acids, and compared the ratios of gene expression in TFL20 with the wild-type strain, K30. We found that the LEU1, LEU2, and BAT1 genes were up-regulated in TFL20 for metabolism, and that TFL20 simultaneously produced as much i-AmOH and leucine as K30 does.

Deciphering cellular functions of protein phosphatases by comparison of gene expression profiles in Saccharomyces cerevisiae

Expression profiles of protein phosphatase (PPase) disruptants were analyzed by use of Pearsons correlation coefficient to find profiles that correlated with those of 316 Reference Gene (RG) disruptants harboring deletions in genes with known functions. Twenty-six Deltappase disruptants exhibited either a positive or negative correlation with 94 RG disruptants when the p value for Pearsons correlation coefficient was >0.2. Some of the predictions that arose from this analysis were tested experimentally and several new Delta ppase phenotypes were found. Notably, Delta sit4 and Delta siw14 disruptants exhibited hygromycin B sensitivity, Delta sit4 and Delta ptc1 disruptants grew slowly on glycerol medium, the Delta ptc1 disruptant was found to be sensitive to calcofluor white and congo red, while the Delta ppg1 disruptant was found to be sensitive to congo red. Because on-going analysis of expression profiles of Saccharomyces cerevisiae disruptants is rapidly generating new data, we suggest that the approach used in the present study to explore PPase function is also applicable to other genes.

Identification and Classification of a Two-Component System Based on Domain Structures in Bacteria and Differences in Domain Structure between Gram-Positive and Gram-Negative Bacteria

Genome sequencing has revealed many pairs of proteins termed two-component systems (TCSs) in bacteria. Each pair consists of a sensor or histidine kinase (HK) and an effector or response regulator (RR). The HK is usually a membrane-spanning protein that senses specific environmental parameters and communicates this information to the cytoplasmic RR protein through phosphotransfer reactions to cope with a variety of environmental stresses, including osmotic pressure, nitrogen lack, phosphoric acid lack, and the presence of oxygen. Furthermore, some proteins have been identified as hybrid kinases composed of HK and RR. We identified the domain structures of 360 bacteria and 43 archaea by domain search against the PFAM database using HMMER. We then classified 8,573 HK, 10,807 RR, and 2,477 hybrid kinases. In addition, we identified specific domains among phylogenic clusters based on differences in domain structure of TCS genes applying the Signal-to-Noise ratio.

Transcriptomic features associated with energy production in the muscles of Pacific bluefin tuna and Pacific cod

Bluefin tuna are high-performance swimmers and top predators in the open ocean. Their swimming is grounded by unique features including an exceptional glycolytic potential in white muscle, which is supported by high enzymatic activities. Here we performed high-throughput RNA sequencing (RNA-Seq) in muscles of the Pacific bluefin tuna (Thunnus orientalis) and Pacific cod (Gadus macrocephalus) and conducted a comparative transcriptomic analysis of genes related to energy production. We found that the total expression of glycolytic genes was much higher in the white muscle of tuna than in the other muscles, and that the expression of only six genes for glycolytic enzymes accounted for 83.4% of the total. These expression patterns were in good agreement with the patterns of enzyme activity previously reported. The findings suggest that the mRNA expression of glycolytic genes may contribute directly to the enzymatic activities in the muscles of tuna. Graphical abstract Comparisons of the rate of total expression levels of genes involved in each of three energy-generating pathways.