Protein structural influences in rhodopsin evolution

Incorporating specific structural information can be important for developing a realistic model of evolution for phylogenetic reconstruction of protein-coding genes. We analyzed 62 sequences of vertebrate rhodopsin. The bovine rhodopsin structure was used to label residue sites by surface accessibility, secondary structure, and transmembrane (TM) location. Residue sites with amino acid differences were identified; using maximum parsimony (MP), homoplasious residues were identified. Residues were analyzed for patterns that would indicate correlation of rate with secondary structure, surface accessibility, or position relative to the lipid bilayer. Surface residues, especially those residing in one of the seven TM helices, were significantly correlated with high rates of amino acid substitution. This category of residues, defined solely by protein structural characteristics, potentially defined a class enriched in homoplasious residues. MP analysis using all sites led to a tree with anomalies in the relationships of amphibian, mammalian, bird, and alligator species. Analysis excluding the structurally defined residue class recovered a more accurate phylogeny. A model is presented for including structural influences on rate in phylogenetic inference.     

十字花科植物PEBP基因家族的分子进化

近年来植物基因组测序物种数量的指数增长, 为我们对植物环境适应性状的遗传和变异的全面理解提供了保障。磷脂酰乙醇胺结合蛋白(phosphatidylethanolamine-binding protein, PEBP)在植物的开花转变和株型建立中起着重要作用, 一直是植物生物学研究关注的热点领域之一。然而对该家族并没有利用新近测序的基因组数据进行比较基因组分析, 制约了对其在分子水平上的进化研究。为了确定PEBP基因家族的分子进化机制, 本研究利用生物信息学方法开展了7种十字花科植物拟南芥(Arabidopsis thaliana)、琴叶拟南芥(A. lyrata)、小鼠耳芥(A. pumila)、亚麻荠(Camelina sativa)、甘蓝(Brassica oleracea)、白菜(B. rapa)和油菜(B. napus)的PEBP基因家族成员的全基因组鉴定、结构特征和比较进化分析。从7个物种中共鉴定出91个PEBP基因, 系统进化分析表明它们分属5个亚家族: MFT、FT/TSF、TFL1、CEN和BFT。基因结构分析发现甘蓝、白菜和油菜的CEN基因内含子明显比其余4个物种的内含子长。蛋白结构域分析表明MFT比其他4个亚家族成员少了一个motif 2, TFL1比其他亚家族多了motif 8。选择压力分析发现7个物种PEBP同源基因均受到较强的纯化选择, 其中TFL1亚家族受到的纯化选择最弱。共线性分析表明十字花科植物PEBP基因家族随古代多倍体事件发生不同程度的扩张, TSF在甘蓝、白菜和油菜中丢失。非生物胁迫下, 在拟南芥中过量表达小鼠耳芥的一个MFT基因, 转基因拟南芥种子的萌发率明显低于野生型, 暗示MFT基因在调控种子萌发上的功能保守。本研究为深入研究十字花科植物PEBP基因的进化特征和生物学功能奠定了基础。     

Identification and molecular evolution of cow CENP-A gene family

The centromere protein A (CENP-A), a histone H3-like protein, provides an essential role for chromosomal segregation during mitosis and meiosis. In this study we identified ten new CENP-A-like genes (excluding the original CENP-A gene) in cow by searching its genome database, while all other examined mammals contained only a single copy of the CENP-A gene. Phylogenetic analysis shows that the expansion of these genes occurred before the divergence of cow and sheep but after the artiodactyls diverged from other mammals. Our analyses also indicate that multiple gene duplication and intron loss events have occurred during the formation of this gene family, and the high similarity in intron sequences between the new genes and the old one suggests very recent gene expansion events. Furthermore, evidence from the comparisons of the synonymous and nonsynonymous substitutions and the distributions of the mutation sites suggested that the CENP-A-L-1 gene, a potentially functional member of cow CENP-A gene family, evolved under positive selection. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome-wide and molecular evolution analysis of the subtilase gene family in Vitis vinifera

Background

Vitis vinifera (grape) is one of the most economically significant fruit crops in the world. The availability of the recently released grape genome sequence offers an opportunity to identify and analyze some important gene families in this species. Subtilases are a group of subtilisin-like serine proteases that are involved in many biological processes in plants. However, no comprehensive study incorporating phylogeny, chromosomal location and gene duplication, gene organization, functional divergence, selective pressure and expression profiling has been reported so far for the grape.

Results

In the present study, a comprehensive analysis of the subtilase gene family in V. vinifera was performed. Eighty subtilase genes were identified. Phylogenetic analyses indicated that these subtilase genes comprised eight groups. The gene organization is considerably conserved among the groups. Distribution of the subtilase genes is non-random across the chromosomes. A high proportion of these genes are preferentially clustered, indicating that tandem duplications may have contributed significantly to the expansion of the subtilase gene family. Analyses of divergence and adaptive evolution show that while purifying selection may have been the main force driving the evolution of grape subtilases, some of the critical sites responsible for the divergence may have been under positive selection. Further analyses of real-time PCR data suggested that many subtilase genes might be important in the stress response and functional development of plants.

Conclusions

Tandem duplications as well as purifying and positive selections have contributed to the functional divergence of subtilase genes in V. vinifera. The data may contribute to a better understanding of the grape subtilase gene family.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1116) contains supplementary material, which is available to authorized users.     

Identification, distribution and molecular evolution of the pacifastin gene family in Metazoa

Background

This study was motivated by the observation of unusual mitochondrial haplotype distributions and associated physiological differences between populations of the killifish Fundulus heteroclitus distributed along the Atlantic coast of North America. A distinct "northern" haplotype is fixed in all populations north of New Jersey, and does not appear south of New Jersey except in extreme upper-estuary fresh water habitats, and northern individuals are known to be more tolerant of hyposmotic conditions than southern individuals. Complete mitochondrial genomes were sequenced from individuals from northern coastal, southern coastal, and fresh water populations (and from out-groups). Comparative genomics approaches were used to test multiple evolutionary hypotheses proposed to explain among-population genome variation including directional selection and hybridization.

Results

Structure and organization of the Fundulus mitochondrial genome is typical of animals, yet subtle differences in substitution patterns exist among populations. No signals of directional selection or hybridization were detected. Mitochondrial genes evolve at variable rates, but all genes exhibit very low dN/dS ratios across all lineages, and the southern population harbors more synonymous polymorphism than other populations.

Conclusion

Evolution of mitochondrial genomes within Fundulus is primarily governed by interaction between strong purifying selection and demographic influences, including larger historical population size in the south. Though directional selection and hybridization hypotheses were not supported, adaptive processes may indirectly contribute to partitioning of variation between populations.     

Identification, distribution and molecular evolution of the pacifastin gene family in Metazoa

Background  

Members of the pacifastin family are serine peptidase inhibitors, most of which are produced as multi domain precursor proteins. Structural and biochemical characteristics of insect pacifastin-like peptides have been studied intensively, but only one inhibitor has been functionally characterised. Recent sequencing projects of metazoan genomes have created an unprecedented opportunity to explore the distribution, evolution and functional diversification of pacifastin genes in the animal kingdom.     

Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family

Summary Sequences of 47 members of the Zn-containing alcohol dehydrogenase (ADH) family were aligned progressively, and an evolutionary tree with detailed branch order and branch lengths was produced. The alignment shows that only 9 amino acid residues (of 374 in the horse liver ADH sequence) are conserved in this family; these include eight Gly and one Val with structural roles. Three residues that bind the catalytic Zn and modulate its electrostatic environment are conserved in 45 members. Asp 223, which determines specificity for NAD, is found in all but the two NADP-dependent enzymes, which have Gly or Ala. Ser or Thr 48, which makes a hydrogen bond to the substrate, is present in 46 members. The four Cys ligands for the structural zinc are conserved except in -crystallin, the sorbitol dehydrogenases, and two bacterial enzymes. Analysis of the evolutionary tree gives estimates of the times of divergence for different animal ADHs. The human class II () and class III () ADHs probably diverged about 630 million years ago, and the newly identified human ADH6 appeared about 520 million years ago, implying that these classes of enzymes may exist or have existed in all vertebrates. The human class I ADH isoenzymes (, , and ) diverged about 80 million years ago, suggesting that these isoenzymes may exist or have existed in all primates. Analysis of branch lengths shows that these plant ADHs are more conserved than the animal ones and that class III ADHs are more conserved than class I ADHs. The rate of acceptance of point mutations (PAM units) shows that selection pressure has existed for ADHs, implying that these enzymes play definite metabolic roles.Offprint requests to: B.V. Plapp     

Comparative genomics in the grass family: molecular characterization of grass genome structure and evolution

The genomes of grasses are very different in terms of size, ploidy level and chromosome number. Despite these significant differences, it was found by comparative mapping that the linear order (colinearity) of genetic markers and genes is very well conserved between different grass genomes. The potential of such conservation has been exploited in several directions, e.g. in defining rice as a model genome for grasses and in designing better strategies for positional cloning in large genomes. Recently, the development of large insert libraries in species such as maize, rice, barley and diploid wheat has allowed the study of large stretches of DNA sequence and has provided insight into gene organization in grasses. It was found that genes are not distributed randomly along the chromosomes and that there are clusters of high gene density in species with large genomes. Comparative analysis performed at the DNA sequence level has demonstrated that colinearity between the grass genomes is retained at the molecular level (microcolinearity) in most cases. However, detailed analysis has also revealed a number of exceptions to microcolinearity, which have given insight into mechanisms that are involved in grass-genome evolution. In some cases, the use of rice as a model to support gene isolation from other grass genomes will be complicated by local rearrangements. In this Botanical Briefing, we present recent progress and future prospects of comparative genomics in grasses.     

Genome-wide and molecular evolution analyses of the phospholipase D gene family in Poplar and Grape

Background  

The Phospholipase D (PLD) family plays an important role in the regulation of cellular processes in plants, including abscisic acid signaling, programmed cell death, root hair patterning, root growth, freezing tolerance and other stress responses. PLD genes constitute an important gene family in higher plants. However, until now our knowledge concerning the PLD gene family members and their evolutionary relationship in woody plants such as Poplar and Grape has been limited.     

Fractal landscapes and molecular evolution: modeling the myosin heavy chain gene family.

Mapping nucleotide sequences onto a "DNA walk" produces a novel representation of DNA that can then be studied quantitatively using techniques derived from fractal landscape analysis. We used this method to analyze 11 complete genomic and cDNA myosin heavy chain (MHC) sequences belonging to 8 different species. Our analysis suggests an increase in fractal complexity for MHC genes with evolution with vertebrate > invertebrate > yeast. The increase in complexity is measured by the presence of long-range power-law correlations, which are quantified by the scaling exponent alpha. We develop a simple iterative model, based on known properties of polymeric sequences, that generates long-range nucleotide correlations from an initially noncorrelated coding region. This new model-as well as the DNA walk analysis-both support the intron-late theory of gene evolution.     

Sorghum phosphoenolpyruvate carboxylase gene family: structure,function and molecular evolution

Although housekeeping functions have been shown for the phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) in plants and in prokaryotes, PEPC is mainly known for its specific role in the primary photosynthetic CO₂ fixation in C4 and CAM plants. We have shown that in Sorghum, a monocotyledonous C4 plant, the enzyme is encoded in the nucleus by a small multigene family. Here we report the entire nucleotide sequence (7.5 kb) of the third member (CP21) that completes the structure of the Sorghum PEPC gene family. Nucleotide composition, CpG islands and GC content of the three Sorghum PEPC genes are analysed with respect to their possible implications in the regulation of expression. A study of structure/function and phylogenetic relationships based on the compilation of all PEPC sequences known so far is presented. Data demonstrate that (1) the different forms of plant PEPC have very similar primary structures, functional and regulatory properties, (2) neither apparent amino acid sequences nor phylogenetic relationships are specific for the C4 and CAM PEPCs and (3) expression of the different genes coding for the Sorghum PEPC isoenzymes is differently regulated (i.e. by light, nitrogen source) in a spatial and temporal manner. These results suggest that the main distinguishing feature between plant PEPCs is to be found at the level of genes expression rather than in their primary structure.     

The molecular weight of rhodopsin and the nature of the rhodopsin-digitonin complex

The sedimentation behavior of aqueous solutions of digitonin and of cattle rhodopsin in digitonin has been examined in the ultracentrifuge. In confirmation of earlier work, digitonin was found to sediment as a micelle (D-1) with an s₂₀ of about 6.35 Svedberg units, and containing at least 60 molecules. The rhodopsin solutions sediment as a stoichiometric complex of rhodopsin with digitonin (RD-1) with an s₂₀ of about 9.77 Svedberg units. The s₂₀ of the RD-1 micelle is constant between pH 6.3 and 9.6, and in the presence of excess digitonin. RD-1 travels as a single boundary also in the electrophoresis apparatus at pH 8.5, and on filter paper at pH 8.0. The molecular weight of the RD-1 micelle lies between 260,000 and 290,000. Of this, only about 40,000 gm. are due to rhodopsin; the rest is digitonin (180 to 200 moles). Comparison of the relative concentrations of RD-1 and retinene in solutions of rhodopsin-digitonin shows that RD-1 contains only one retinene equivalent. It can therefore contain only one molecule of rhodopsin with a molecular weight of about 40,000. Cattle rhodopsin therefore contains only one chromophore consisting of a single molecule of retinene. It is likely that frog rhodopsin has a similar molecular weight and also contains only one chromophore per molecule. The molar extinction coefficient of rhodopsin is therefore identical with the extinction coefficient per mole of retinene (40,600 cm.² per mole) and the E(1 per cent, 1 cm., 500 mµ) has a value of about 10. Rhodopsin constitutes about 14 per cent of the dry weight, and 3.7 per cent of the wet weight of cattle outer limbs. This corresponds to about 4.2 x 10⁶ molecules of rhodopsin per outer limb. The rhodopsin content of frog outer limbs is considerably higher: about 35 per cent of the dry weight, and 10 per cent of the wet weight, corresponding to about 2.1 x 10⁹ molecules per outer limb. Thus the frog outer limb contains about five hundred times as much rhodopsin as the cattle outer limb. But the relative volumes of these structures are such that the ratio of concentrations is only about 2.5 to 1 on a weight basis. Rhodopsin accounts for at least one-fifth of the total protein of the cattle outer limb; for the frog, this value must be higher. The extinction (K₅₀₀) along its axis is about 0.037 cm.² for the cattle outer limb, and about 0.50 cm.² for the frog outer limb.     

Expansion and molecular evolution of the interferon-induced 2'-5' oligoadenylate synthetase gene family

The mammalian 2'-5' oligoadenylate synthetases (2'-5'OASs) are enzymes that are crucial in the interferon-induced antiviral response. They catalyze the polymerization of ATP into 2'-5'-linked oligoadenylates which activate a constitutively expressed latent endonuclease, RNaseL, to block viral replication at the level of mRNA degradation. A molecular evolutionary analysis of available OAS sequences suggests that the vertebrate genes are members of a multigene family with its roots in the early history of tetrapods. The modern mammalian 2'-5'OAS genes underwent successive gene duplication events resulting in three size classes of enzymes, containing one, two, or three homologous domains. Expansion of the OAS gene family occurred by whole-gene duplications to increase gene content and by domain couplings to produce the multidomain genes. Evolutionary analyses show that the 2'-5'OAS genes in rodents underwent gene duplications as recently as 11 MYA and predict the existence of additional undiscovered OAS genes in mammals.     

Molecular evolution of the rhodopsin gene of marine lamprey,Petromyzon marinus

Visual pigment genes have been isolated from a marine lamprey, Petromyzon marinus. We report here the rhodopsin gene, spanning 21.2 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Southern analysis suggests that the lamprey genome contains a single rhodopsin gene. The amino acid (aa) sequence deduced from this gene has 92% sequence similarity with that of the river lamprey rhodopsin. The data reveal that aa substitutions occurred more often in the transmembrane region than in the non-transmembrane region, possibly reflecting functional adaptation of the rhodopsin during the last 500 million years of the jawless fish evolution.     

Detecting molecular interactions that stabilize native bovine rhodopsin

Using single-molecule force spectroscopy we probed molecular interactions within native bovine rhodopsin and discovered structural segments of well-defined mechanical stability. Highly conserved residues among G protein-coupled receptors were located at the interior of individual structural segments, suggesting a dual role for these segments in rhodopsin. Firstly, structural segments stabilize secondary structure elements of the native protein, and secondly, they position and hold the highly conserved residues at functionally important environments. Two main classes of force curves were observed. One class corresponded to the unfolding of rhodopsin with the highly conserved Cys110-Cys187 disulfide bond remaining intact and the other class corresponded to the unfolding of the entire rhodopsin polypeptide chain. In the absence of the Cys110-Cys187 bond, the nature of certain molecular interactions within folded rhodopsin was altered. These changes highlight the structural importance of this disulfide bond and may form the basis of dysfunctions associated with its absence.     

Conservation of molecular interactions stabilizing bovine and mouse rhodopsin

Rhodopsin is the light receptor that initiates phototransduction in rod photoreceptor cells. The structure and function of rhodopsin are tightly linked to molecular interactions that stabilize and determine the receptor's functional state. Single-molecule force spectroscopy (SMFS) was used to localize and quantify molecular interactions that structurally stabilize bovine and mouse rhodopsin from native disk membranes of rod photoreceptor cells. The mechanical unfolding of bovine and mouse rhodopsin revealed nine major unfolding intermediates, each intermediate defining a structurally stable segment in the receptor. These stable structural segments had similar localization and occurrence in both bovine and mouse samples. For each structural segment, parameters describing their unfolding energy barrier were determined by dynamic SMFS. No major differences were observed between bovine and mouse rhodopsin, thereby implying that the structures of both rhodopsins are largely stabilized by similar molecular interactions.     

Primary structure of frog PYY: implications for the molecular evolution of the pancreatic polypeptide family.

A peptide belonging to the pancreatic polypeptide (PP) family was isolated in pure form from the intestine of the European green frog (Rana ridibunda). The primary structure of the peptide was established as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu10-Asp-Ala- Ser-Pro-Glu-Glu-Met-Thr-Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile- Asn-Leu30-Val - Thr-Arg-Gln-Arg-Tyr-NH2. This amino acid sequence shows moderate structural similarity to human PYY (75% identity) but stronger similarity to the PP family peptides isolated from the pancreas of the salmon (86%) and dogfish (83%). The data suggest that the two putative duplications of an ancestral PP family gene that have given rise to PP, PYY and NPY in mammals had already taken place by the time of the appearance of the amphibia. In fish, however, only a single duplication has occurred, giving rise to NPY in nervous tissue and a PYY-related peptide in both pancreas and gut.     

An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.