On the Structural Differences Between Markers and Genomic AC Microsatellites

AC microsatellites have proved particularly useful as genetic markers. For some purposes, such as in population biology, the inferences drawn depend on the quantitative values of their mutation rates. This, together with intrinsic biological interest, has led to widespread study of microsatellite mutational mechanisms. Now, however, inconsistencies are appearing in the results of marker-based versus non-marker-based studies of mutational mechanisms. The reasons for this have not been investigated, but one possibility, pursued here, is that the differences result from structural differences between markers and genomic microsatellites. Here we report a comparison between the CEPH AC marker microsatellites and the global population of AC microsatellites in the human genome. AC marker microsatellites are longer than the global average. Controlling for length, marker microsatellites contain on average fewer interruptions, and have longer segments, than their genomic counterparts. Related to this, marker microsatellites show a greater tendency to concentrate the majority of their repeats into one segment. These differences plausibly result from scientists selecting markers for their high polymorphism. In addition to the structural differences, there are differences in the base composition of flanking sequences, marker flanking regions being richer in C and G and poorer in A and T. Our results indicate that there are profound differences between marker and genomic microsatellites that almost certainly affect their mutation rates. There is a need for a unified model of mutational mechanisms that accounts for both marker-derived and genomic observations. A suggestion is made as to how this might be done.Reviewing Editor: Dr. Magnto Nordborg     

稻瘟菌WD重复功能域中SSR的变异及其对蛋白结构的影响

含WD重复功能域的蛋白能够参与信号传导、转录调控、RNA剪切、细胞的凋亡等多种功能,在病原菌与寄主植物蛋白互作的过程中扮演着重要的角色。本研究分析了稻瘟病菌基因组中94个WD功能域基因编码区和调控区中SSR的组成、分布,并检测了7个蛋白编码区中SSR的变异及其对蛋白二级结构的影响。结果表明,WD功能域基因的编码区和调控区中都含有大量的SSR,但是SSR在这些基因的外显子区、内含子区、5’一UTR和3’一UTR区中SSR的组成和分布均不相同;编码区中三碱基和六碱基SSR分布较多,这些SSR基序大都表现为GC含量较高和其所编码的亲水性氨基酸出现的频率远远高于疏水性氨基酸的特点。且检测的7个WD功能域基因的编码区中的SSR位点均具有丰富的多态性,通过Antheprot（DPM）软件预测发现：SSR的变异对蛋白的二级结构有一定影响。这暗示着SSR的变异对致病相关基因的变异起着十分重要的作用。     

蛋白质结构与功能中的结构域

结构域是蛋白质亚基结构中的紧密球状区域.结构域作为蛋白质结构中介于二级与三级结构之间的又一结构层次,在蛋白质中起着独立的结构单位、功能单位与折叠单位的作用.在复杂蛋白质中,结构域具有结构与功能组件与遗传单位的作用.结构域层次的研究将会促进蛋白质结构与功能关系、蛋白质折叠机制以及蛋白质设计的研究.     

The Comparative Genomics of Polyglutamine Repeats: Extreme Difference in the Codon Organization of Repeat-Encoding Regions Between Mammals and Drosophila

Polyglutamine repeats within proteins are common in eukaryotes and are associated with neurological diseases in humans. Many are encoded by tandem repeats of the codon CAG that are likely to mutate primarily by replication slippage. However, a recent study in the yeast Saccharomyces cerevisiae has indicated that many others are encoded by mixtures of CAG and CAA which are less likely to undergo slippage. Here we attempt to estimate the proportions of polyglutamine repeats encoded by slippage-prone structures in species currently the subject of genome sequencing projects. We find a general excess over random expectation of polyglutamine repeats encoded by tandem repeats of codons. We nevertheless find many repeats encoded by nontandem codon structures. Mammals and Drosophila display extreme opposite patterns. Drosophila contains many proteins with polyglutamine tracts but these are generally encoded by interrupted structures. These structures may have been selected to be resistant to slippage. In contrast, mammals (humans and mice) have a high proportion of proteins in which repeats are encoded by tandem codon structures. In humans, these include most of the triplet expansion disease genes. Received: 17 August 2000 / Accepted: 20 November 2000     

QAUST: Protein Function Prediction Using Structure Similarity,Protein Interaction,and Functional Motifs

The number of available protein sequences in public databases is increasing exponentially. However, a significant percentage of these sequences lack functional annotation, which is essential for the understanding of how biological systems operate. Here, we propose a novel method, Quantitative Annotation of Unknown STructure (QAUST), to infer protein functions, specifically Gene Ontology (GO) terms and Enzyme Commission (EC) numbers. QAUST uses three sources of information: structure information encoded by global and local structure similarity search, biological network information inferred by protein–protein interaction data, and sequence information extracted from functionally discriminative sequence motifs. These three pieces of information are combined by consensus averaging to make the final prediction. Our approach has been tested on 500 protein targets from the Critical Assessment of Functional Annotation (CAFA) benchmark set. The results show that our method provides accurate functional annotation and outperforms other prediction methods based on sequence similarity search or threading. We further demonstrate that a previously unknown function of human tripartite motif-containing 22 (TRIM22) protein predicted by QAUST can be experimentally validated.     

Protein Superfamily Evolution and the Last Universal Common Ancestor (LUCA)

By exploiting three-dimensional structure comparison, which is more sensitive than conventional sequence-based methods for detecting remote homology, we have identified a set of 140 ancestral protein domains using very restrictive criteria to minimize the potential error introduced by horizontal gene transfer. These domains are highly likely to have been present in the Last Universal Common Ancestor (LUCA) based on their universality in almost all of 114 completed prokaryotic (Bacteria and Archaea) and eukaryotic genomes. Functional analysis of these ancestral domains reveals a genetically complex LUCA with practically all the essential functional systems present in extant organisms, supporting the theory that life achieved its modern cellular status much before the main kingdom separation (Doolittle 2000). In addition, we have calculated different estimations of the genetic and functional versatility of all the superfamilies and functional groups in the prokaryote subsample. These estimations reveal that some ancestral superfamilies have been more versatile than others during evolution allowing more genetic and functional variation. Furthermore, the differences in genetic versatility between protein families are more attributable to their functional nature rather than the time that they have been evolving. These differences in tolerance to mutation suggest that some protein families have eroded their phylogenetic signal faster than others, hiding in many cases, their ancestral origin and suggesting that the calculation of 140 ancestral domains is probably an underestimate. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zarobya]     

Ligand Binding Site Structure Influences the Evolution of Protein Complex Function and Topology

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Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.     

Reticulate Evolution of Parthenospecies of the Lacertidae Rock Lizards: Inheritance of CLsat Tandem Repeats and Anonymous RAPD Markers

The genetic relatedness of several bisexual and of four unisexual Lacerta saxicola complex lizards was studied, using monomer sequences of the complex-specific CLsat tandem repeats and anonymous RAPD markers. Genomes of parthenospecies were shown to include different satellite monomers. The structure of each such monomer is specific for a certain pair of bisexual species. This fact might be interpreted in favor of co-dominant inheritance of these markers in bisexual species hybridogenesis. This idea is supported by the results obtained with RAPD markers; i.e., unisexual species genomes include only the loci characteristic of certain bisexual species. At the same time, in neither case parthenospecies possess specific, autoapomorphic loci that were not present in this or that bisexual species.     

The Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (M_r = ∼3.5 million), assembled from 10 identical copies of an ∼350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of ∼50 kDa each (FUs a–g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5′ UTR of 58 bp, a 3′ UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (M_r = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin “OdH” from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 ± 24 million years ago, in close agreement with fossil records from the early Devonian. [Reviewing Editor: Dr. Axel Meyer] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ619741.     

Testing for Spatial Clustering of Amino Acid Replacements Within Protein Tertiary Structure

Widely used models of protein evolution ignore protein structure. Therefore, these models do not predict spatial clustering of amino acid replacements with respect to tertiary structure. One formal and biologically implausible possibility is that there is no tendency for amino acid replacements to be spatially clustered during evolution. An alternative to this is that amino acid replacements are spatially clustered and this spatial clustering can be fully explained by a tendency for similar rates of amino acid replacement at sites that are nearby in protein tertiary structure. A third possibility is that the amount of clustering exceeds that which can be explained solely on the basis of independently evolving protein sites with spatially clustered replacement rates. We introduce two simple and not very parametric hypothesis tests that help distinguish these three possibilities. We then apply these tests to 273 homologous protein families. The null hypothesis of no spatial clustering is rejected for 102 of 273 families. The explanation of spatially clustered rates but independent change among sites is rejected for 43 families. These findings need to be reconciled with the common practice of basing evolutionary inferences on models that assume independent change among sites. [Reviewing Editior: Dr. David Pollock]     

A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties

Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.     

后基因组研究中蛋白结构与功能的预测

阐述蛋白质结构建模和功能预测的基本方法以及最新研究进展,展望了蛋白质预测技术的前景。     

Evolution of keratin genes: Different protein domains evolve by different pathways

Summary Intermediate filaments are composed of a family of proteins that evolved from a common ancestor. The proteins consist of three domains: a central, alpha-helical domain similar in all intermediate filaments, bracketed by two domains that are variable in length and structure. Within the intermediate-filament family, several subfamilies have been recognized by immunologic and nucleic acid hybridization techniques. In this paper we present the sequence of the genomic DNA coding for a 65-kilodalton human keratin and compare it with the sequences of other intermediate-filament proteins. While the central, alpha-helical domains of these proteins show homologies that indicate a common ancestor, the sequences of the variable terminal domains indicate that the variable domains evolved through a series of tandem duplications and possibly by gene-conversion mechanisms.     

On the Classification and Evolution of Protein Modules

Our efforts to classify the functional units of many proteins, the modules, are reviewed. The data from the sequencing projects for various model organisms are extremely helpful in deducing the evolution of proteins and modules. For example, a dramatic increase of modular proteins can be observed from yeast to C. elegans in accordance with new protein functions that had to be introduced in multicellular organisms. Our sequence characterization of modules relies on sensitive similarity search algorithms and the collection of multiple sequence alignments for each module. To trace the evolution of modules and to further automate the classification, we have developed a sequence and a module alerting system that checks newly arriving sequence data for the presence of already classified modules. Using these systems, we were able to identify an unexpected similarity between extracellular C1Q modules with bacterial proteins.     

Evolution,Mechanisms, and Applications of Intein-mediated Protein Splicing

Intein-mediated protein splicing raises questions and creates opportunities in many scientific areas. Evolutionary biologists question whether inteins are primordial enzymes or simply selfish elements, whereas biochemists seek to understand how inteins work. Synthetic chemists exploit inteins in the semisynthesis of proteins with or without nonribosomal modifications, whereas biotechnologists use modified inteins in an ever increasing variety of applications. The four minireviews in this series explore these themes. The first minireview focuses on the evolution and biological function of inteins, whereas the second describes the mechanisms that underlie the remarkable ability of inteins to perform complex sets of choreographed enzymatic reactions. The third explores the relationship between the three-dimensional structure and dynamics of inteins and their biochemical capabilities. The fourth describes intein applications that have moved beyond simple technology development to utilizing inteins in more sophisticated applications, such as biosensors for identifying ligands of human hormone receptors or improved methods of biofuel and plant-based sugar production.     

Comparative mechanical unfolding studies of spectrin domains R15, R16 and R17

Spectrins belong to repetitive three-helix bundle proteins that have vital functions in multicellular organisms and are of potential value in nanotechnology. To reveal the unique physical features of repeat proteins we have studied the structural and mechanical properties of three repeats of chicken brain α-spectrin (R15, R16 and R17) at the atomic level under stretching at constant velocities (0.01, 0.05 and 0.1?Å·ps^?1) and constant forces (700 and 900?pN) using molecular dynamics (MD) simulations at T?=?300?K. 114 independent MD simulations were performed and their analysis has been done. Despite structural similarity of these domains we have found that R15 is less mechanically stable than R16, which is less stable than R17. This result is in agreement with the thermal unfolding rates. Moreover, we have observed the relationship between mechanical stability, flexibility of the domains and the number of aromatic residues involved in aromatic clusters.     

Protein and Genome Evolution in Mammalian Cells for Biotechnology Applications

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.