拟南芥基因组中注释为小檗碱桥环酶基因的分子克隆及异源表达

小檗碱桥环酶(BBE)催化(S)-牛心果碱((S)-reticuline)中N-CH3与分子内苄基部分中羟基的邻位芳香碳之间C-C键的形成,该酶属于双共价黄素蛋白家族,是苄基异喹啉类生物碱向小檗碱类生物碱转化的关键酶.迄今,在拟南芥(Arabidopsis thaliana)中尚未发现复杂生物碱,但其基因组测序结果表明拟南芥含有众多可能与复杂生物碱生物合成相关的基因,其中与BBE类似的基因有12个.基于与已知功能的BBE及拟南芥中BBE序列的分析,选定拟南芥中4个注释为BBE的编码基因为目的基因,设计特异引物,从拟南芥cDNA中扩增并克隆到pGM-T载体中,筛选重组子,测序并分析,获得了4个BBE目的基因,分别为AT2G34810、AT5G44400、AT5G44410和AT5G44440.将上述基因克隆至表达载体pET-28a或pET-30a中,分别转入大肠杆菌Rosetta(DE3)中,IPTG诱导实现了上述基因的异源表达.     

Peptide synthesis through evolution

Ribosome-catalyzed peptide bond formation is a crucial function of all organisms. The ribosome is a ribonucleoprotein particle, with both RNA and protein components necessary for the various steps leading to protein biosynthesis. Evolutionary theory predicts an early environment devoid of complex biomolecules, and prebiotic peptide synthesis would have started in a simple way. A fundamental question regarding peptide synthesis is how the current ribosome-catalyzed reaction evolved from a primitive system. Here we look at both prebiotic and modern mechanisms of peptide bond formation and discuss recent experiments that aim to connect these activities. In particular, RNA can facilitate peptide bond formation by providing a template for activated amino acids to react and can catalyze a variety of functions that would have been necessary in a pre-protein world. Therefore, RNA may have facilitated the emergence of the current protein world from an RNA or even prebiotic world.Received 4 December 2003; received after revision 13 January 2004; accepted 15 January 2004     

Organization and expression of the poxvirus genome

Summary Poxviruses comprise a large group of very complex animal DNA viruses which replicate in the cytoplasm of infected cells. Vaccinia virus, the most studied poxvirus, has a linear, double stranded DNA genome with an approximate molecular weight of 120×10⁶ (180 kilobase pairs). The two strands of the DNA molecule are naturally cross-linked at both termini. In addition, the vaccinia virus genome contains very long inverted terminal repetitions of approximately 10 kilobase pairs which are further characterized by the presence of direct tandem repeats of a 70-base-pair sequence arranged in two blocks of 13 and 17 copies, respectively. A central region of the genome is highly conserved between different orthopoxviruses. In contrast, the ends are hypervariable and may contain extensive deletions and complex, symmetrical sequences rearrangements. Vaccinia virus gene expression is divided into two stages. Early in infection, RNA complementary to one half of one strand-equivalent of the genome is transcribed within subviral particles by the virion-associated RNA polymerase. Later in infection, after DNA replication, RNA complementary to one entire strand-equivalent is transcribed. RNA made late in infection is very heterogeneous in length and a large fraction of it contains self-complementary sequences. Late genes are clustered near the central region of the genome. Vaccinia virus mRNAs do not appear to be synthesized by a splicing mechanism.     

Current topics in genome evolution: Molecular mechanisms of new gene formation

Comparative genome analyses reveal that most functional domains of human genes have homologs in widely divergent species. These shared functional domains, however, are differentially shuffled among evolutionary lineages to produce an increasing number of domain architectures. Combined with duplication and adaptive evolution, domain shuffling is responsible for the great phenotypic complexity of higher eukaryotes. Although the domain-shuffling hypothesis is generally accepted, determining the molecular mechanisms that lead to domain shuffling and novel gene creation has been challenging, as sequence features accompanying the formation of known genes have been obscured by accumulated mutations. The growing availability of genome sequences and EST databases allows us to study the characteristics of newly emerged genes. Here we review recent genome-wide DNA and EST analyses, and discuss the three major molecular mechanisms of gene formation: (1) atypical spicing, both within and between genes, followed by adaptation, (2) tandem and interspersed segmental duplications, and (3) retrotransposition events. Received 18 October 2006; received after revision 18 November 2006; accepted 28 November 2006