A universal trend of amino acid gain and loss in protein evolution

Amino acid composition of proteins varies substantially between taxa and, thus, can evolve. For example, proteins from organisms with (G + C)-rich (or (A + T)-rich) genomes contain more (or fewer) amino acids encoded by (G + C)-rich codons. However, no universal trends in ongoing changes of amino acid frequencies have been reported. We compared sets of orthologous proteins encoded by triplets of closely related genomes from 15 taxa representing all three domains of life (Bacteria, Archaea and Eukaryota), and used phylogenies to polarize amino acid substitutions. Cys, Met, His, Ser and Phe accrue in at least 14 taxa, whereas Pro, Ala, Glu and Gly are consistently lost. The same nine amino acids are currently accrued or lost in human proteins, as shown by analysis of non-synonymous single-nucleotide polymorphisms. All amino acids with declining frequencies are thought to be among the first incorporated into the genetic code; conversely, all amino acids with increasing frequencies, except Ser, were probably recruited late. Thus, expansion of initially under-represented amino acids, which began over 3,400 million years ago, apparently continues to this day.     

Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo

Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic L-pyrrolysine is attached as a free molecule to tRNA(CUA) by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNA(CUA) in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNA(CUA)) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.     

大肠杆菌基因编码区碱基分布非均匀性的研究

基于大肠杆菌 １２９２ 个基因编码区．１）统计出了氨基酸丰度分布,与一般生物氨基酸丰度比较发现,构成大肠杆菌蛋白质的氨基酸中疏水氨基酸相对较多,小氨基酸相对较少,二硫键相对较少;２）发现编码区碱基分布的非均匀性与氨基酸丰度的分布和同义密码子的非均匀使用紧密相关;３）分析了编码开始区域、编码终止区域碱基分布和氨基酸丰度分布的差别,得到了一些有意义的结论．     

Characterization and expression of a cDNA, AmphiSDHD , encoding the amphioxus cytochrome b small subunit in mitochondrial succinate ubiquinone oxidoreductase

Succinate ubiquinone oxidoreductase ,alsoknownascomplexII ,isthesmallestenzymecomplexfunctioninginboththetricarboxylicacidcycle(TAC)andtheaerobicrespiratorychainofeukaryoticcellmitochondriaandprokaryoticcells[1] .Itiscom posedoffournuclear encoded proteins ,SDHAorflavoprotein (70kD) ,SDHBoriron sulfurprotein(2 7kD) ,SDHCorCII 3(15kD)andSDHDorCII 4 (7— 9kD)inmosteukaryotes[2 ] .Thetwoproteins ,SDHAandSDHB ,comprisethesuccinatedehydroge nase (SDH) ,anenzymeoftheTACandaperipher…     

Characterization and expression of a cDNA, AmphiSDHD,encoding the amphioxus cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase

In this study, an amphioxus cDNA, AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 1429 bp in length, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids rich in basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.     

The codon CUG is read as serine in an asporogenic yeast Candida cylindracea

Deviations from the universal genetic code have been reported for several microorganisms. Termination codons are used for coding some amino acids in Paramecium, Mycoplasma or Tetrahymena, and in Escherichia coli, the UGA termination codon is used to code for selenocysteine. In mitochondria, the changes of sense codons to termination codons or to codons encoding other amino acids have also been reported. Here we report another example of divergence from the universal code, this time in a non-spore-forming yeast Candida cylindracea, in which the universal codon for leucine, CUG, is used to code for serine. This conclusion is based on the observations that: (1) the amino-acid composition and the partial amino-acid sequences of an extracellular lipase from this yeast agreed with those deduced from the complementary DNA if CUG was assumed to specify serine; and (2) serine, but not leucine, was incorporated into a polypeptide in a cell-free translation system from this yeast in the presence of a synthetic CUG oligomer.     

分子序列的突变危险性与遗传密码的编码规则

定义了分子序列（碱基序更和对应的氨基酸序列）的突变危险性函数，通过危险性函数的极小化可以导出遗传密码的简并规则，终止密码子在密码表上的位置及亲－水畴排布，氨基酸在密码表上的定位接近标准密码表，各个生物类别的突变危险性函数值差别不超过５％，可看作守恒量，Ｒａｓ家庭癌基因产物具有较大的突变危险性函数值，这提示分子序列的突变危险性函数可能有重要的应用价值和理论意义。     

Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code.

One serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role.     

C-terminal truncated glucose transporter is locked into an inward-facing form without transport activity

The facilitated glucose transporters comprise a structurally related family of proteins predicted to have 12 membrane-spanning domains, with the amino terminus, a relatively large middle loop and the carboxy-terminus all oriented towards the cytoplasm. An alternating conformation model has been proposed to explain the mechanism of facilitated glucose transport. To understand the structure-function relationships, especially the role of the intracellular C-terminal domain, we have modified the rabbit equivalent of the erythroid-type transporter, GLUT1 (ref. 18), using complementary DNA to code for a deletion mutant that lacks most (37 out of 42 amino acids) of the intracellular C-terminal domain. This deletion mutant is expressed at the cell surface of Chinese hamster ovary (CHO) cells, but is functionally inactive, probably because it has lost its capacity to alternate in conformation and so is locked into an inward-facing form.     

模糊极值与遗传密码的亲水—疏水性和突变危险性

提出了两遗传密切之间距离的一个定义，以该定义为基础构造了密切字典的突变危险度函数；应用模糊极值方法求出了使突变危险度极小的标准密码表的疏水－亲水畴分布；证实由局域极小化的简并多重态构成的标准密码字典和大部分线粒体密码字典的突变危险度是近似极小的，若以密切字典系统平均突变危险度和最大突变危险度为标度，现有的各种与极小值的接近程度在90％左右；计算结果还支持这样的假设：密码的进化是遵循整体突变危险性极小的，标准密码表可能是由类似于线料 密码的古典密码表进行化来的，而碱基的化学修饰有助于降低突变危险性。     

An aminoacyl tRNA synthetase whose sequence fits into neither of the two known classes

Aminoacyl transfer RNA synthetases catalyse the first step of protein synthesis and establish the rules of the genetic code through the aminoacylation of tRNAs. There is a distinct synthetase for each of the 20 amino acids and throughout evolution these enzymes have been divided into two classes of ten enzymes each. These classes are defined by the distinct architectures of their active sites, which are associated with specific and universal sequence motifs. Because the synthesis of aminoacyl-tRNAs containing each of the twenty amino acids is a universally conserved, essential reaction, the absence of a recognizable gene for cysteinyl tRNA synthetase in the genomes of Archae such as Methanococcus jannaschii and Methanobacterium thermoautotrophicum has been difficult to interpret. Here we describe a different cysteinyl-tRNA synthetase from M. jannaschii and Deinococcus radiodurans and its characterization in vitro and in vivo. This protein lacks the characteristic sequence motifs seen in the more than 700 known members of the two canonical classes of tRNA synthetase and may be of ancient origin. The existence of this protein contrasts with proposals that aminoacylation with cysteine in M. jannaschii is an auxiliary function of a canonical prolyl-tRNA synthetase.     

遗传密码突变危险性的全局极小

建立遗传密码稳定性的数学理论,给出遗传密码总体突变危险性的一个数学定义,由此出发讨论它的全局极化化,求出突变危险性全局极小化的密码表,并研究在什么条件下可以导出标准密码表。     

密码子、氨基酸若干重要性质的相关性

对遗传密码子及20种蛋白质氨基酸的若干重要性质的相互关系进行了考察，发现，不仅在密码子和氨基酸内部的多项性质间。而且在密码子与氨基酸两者的多项性质间均存在着显著的相关性．这种相关性在核酸与蛋白质之间逻辑关系的分析方面可能有意义．     

Mimicking the alloantigenicity of proteins with chemically synthesized peptides differing in single amino acids

Recent studies have shown that short chemically synthesized peptides very often induce antibodies which react with the cognate sequence in the intact folded protein. Since such antibodies react with known regions of proteins, they are of predetermined specificity and offer a precision not previously possible with immunological probes. A basic concept emerging from the use of such antibodies in viral systems is that the differential immunogenicity of closely related proteins can be mimicked by short peptides which span the regions of sequence variation. To generalize this concept, we have studied the two Thy-1 proteins which vary by only a single amino acid. Chemically synthesized peptides differing in only one out of 19 amino acids were able to induce allospecific antisera. Thus, single amino acid changes have similar effects on the immunogenicity of proteins and small peptides, even though the latter are free from constraints provided by neighbouring structures in the tertiary configuration of the intact folded proteins.     

Branched chain amino acids and metabolic regulation

Amino acids are fundamental nutrients required for protein synthesis. The branched chain amino acids (BCAAs) leucine, isoleucine, and valine are the most abundant of the essential amino acids. BCAAs have recently been recognized as having functions in processes other than simple nutrition. For example, metabolic diseases are characterized by higher levels of circulating BCAAs. Moreover, supplementation with or deficiency in BCAAs is closely related to the regulation of metabolic homeostasis. Indeed, leucine deprivation induces increased lipolysis and thermogenesis, which result in fat loss, as well as suppressed lipogenesis and enhanced insulin sensitivity in the liver. Accumulating evidence has indicated that several amino acid sensors, including GCN2, ATF4, mTOR, and AMPK, play pivotal roles in the regulation of lipid metabolism, glucose metabolism, and energy homeostasis. Furthermore, the hypothalamus is critical for sensing amino acid levels and mediates the metabolic adaptation of the body upon limitation of essential amino acids (EAAs) through regulating expression of the S6K1, MC4R, and CRH. In this review, we highlight recent studies investigating the cellular mechanisms linking amino acids, amino acid sensors, metabolic regulation, and metabolic diseases. Amino acid sensing and metabolic regulation have become research hotspots in the metabolic field.     

The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine

Pyrrolysine, the twenty-second amino acid found to be encoded in the natural genetic code, is necessary for all of the known pathways by which methane is formed from methylamines. Pyrrolysine comprises a methylated pyrroline carboxylate in amide linkage to the ε-amino group of L-lysine. In certain Archaea, three methyltransferases initiate methanogenesis from the various methylamines, and these enzymes are encoded by genes with an in-frame amber codon that is translated as pyrrolysine. Escherichia coli that has been transformed with the pylTSBCD genes from methanogenic Archaea can incorporate endogenously biosynthesized pyrrolysine into proteins. The decoding of UAG as pyrrolysine requires pylT, which produces tRNA(Pyl) (also called tRNA(CUA)), and pylS, which encodes a pyrrolysyl-tRNA synthetase. The pylB, pylC and pylD genes are each required for tRNA-independent pyrrolysine synthesis. Pyrrolysine is the last remaining genetically encoded amino acid with an unknown biosynthetic pathway. Here we provide genetic and mass spectrometric evidence for a pylBCD-dependent pathway in which pyrrolysine arises from two lysines. We show that a newly uncovered UAG-encoded amino acid, desmethylpyrrolysine, is made from lysine and exogenous D-ornithine in a pylC-dependent process followed by a pylD-dependent process, but it is not further converted to pyrrolysine. These results indicate that the radical S-adenosyl-L-methionine (SAM) protein PylB mediates a lysine mutase reaction that produces 3-methylornithine, which is then ligated to a second molecule of lysine by PylC before oxidation by PylD results in pyrrolysine. The discovery of lysine as the sole precursor to pyrrolysine will further inform discussions of the evolution of the genetic code and amino acid biosynthetic pathways. Furthermore, intermediates of the pathway may provide new avenues by which the pyl system can be exploited to produce recombinant proteins with useful modified residues.     

Myxococcus xanthus spore coat protein S may have a similar structure to vertebrate lens beta gamma-crystallins

The Gram-negative bacterium Myxococcus xanthus has a complex life cycle during which large amounts of a protein of relative molecular mass (Mr) 19,000, known as protein S, are assembled into a spore surface coat by a process that specifically requires calcium ions. The gene for protein S has been cloned and the DNA sequence shows that the gene product is composed of four internally repeated homologous sequences, each 40 amino acids long. Although protein S resembles calmodulin both in its internally duplicated structure and its ability to bind calcium, it apparently has a beta-sheet secondary structure rather than the helix-loop-helix motifs that characterize the calmodulin family. We now show that protein S has a striking homology with the beta- and gamma-crystallins of the vertebrate eye lens which are beta-sheet proteins with internally duplicated structures. This implies that the beta- and gamma-crystallins evolved from already existing proteins, whose ancestors occurred in the prokaryotes. The biological function of protein S, as a closely packed, stable protein in a relatively dehydrated environment, has implications for the functions of crystallins, which are found closely packed in the lens fibre cells, where their stability is essential for maintenance of transparency.     

基于关系数据模型代码生成器的设计与实现

在以多层架构实现的数据库应用系统中,大量程序代码与其关系数据模型直接相关,采用一种以关系数据模型为输入的自动代码生成工具,可以大幅度地降低编程工作量,提高软件质量.现有基于模板的代码生成工具具有良好的设计框架,但其脚本语言由于与关系数据模型关联不紧密,导致代码模板编写和维护比较困难,难以快速应用.文中设计一种与关系数据模型紧密结合的迭代式代码脚本语言RDM CodeMark,保留基于模板的代码生成技术通用性的同时,简化代码模板编写难度.结果表明:基于RDM CodeMark语言的代码生成工具在针对J2EE平台和.Net平台的软件项目开发实践中能取得较为理想的效果.     

α-氨基酸-甲醇-水三元体系中分子间的异系焓相互作用

探讨α 氨基酸 -甲醇 -水三元体系中溶质分子间相互作用机理 ,为揭示蛋白质的稳定机理、蛋白质变性的原因和生物大分子与小分子间的相互作用提供溶液热力学依据 .利用 2 2 77热活性检测仪的流动测量系统获得了 2 98.1 5K时甘氨酸、L 丙氨酸、和L 脯氨酸分别与甲醇在水溶液中的混合过程焓变以及各自的稀释焓 ,依据McMillan Mayer理论关联得到异系焓相互作用系数 ,讨论了不同氨基酸与甲醇分子的作用机理 .不同氨基酸与甲醇分子间的焓作用系数的大小主要取决于氨基酸分子结构的差异 ,氨基酸的不同侧基 (非极性或极性 )对焓作用系数有着不同的贡献 ,脯氨酸特殊的五元吡咯环结构对hxy 值有较大的正贡献 .