Relative Rates of Nucleotide Substitution in Frogs

Accurate estimation of relative mutation rates of mitochondrial DNA (mtDNA) and single-copy nuclear DNA (scnDNA) within lineages contributes to a general understanding of molecular evolutionary processes and facilitates making demographic inferences from population genetic data. The rate of divergence at synonymous sites (K_s) may be used as a surrogate for mutation rate. Such data are available for few organisms and no amphibians. Relative to mammals and birds, amphibian mtDNA is thought to evolve slowly, and the K_s ratio of mtDNA to scnDNA would be expected to be low as well. Relative K_s was estimated from a mitochondrial gene, ND2, and a nuclear gene, c-myc, using both approximate and likelihood methods. Three lineages of congeneric frogs were studied and this ratio was found to be approximately 16, the highest of previously reported ratios. No evidence of a low K_s in the nuclear gene was found: c-myc codon usage was not biased, the K_s was double the intron divergence rate, and the absolute K_s was similar to estimates obtained here for other genes from other frog species. A high K_s in mitochondrial vs. nuclear genes was unexpected in light of previous reports of a slow rate of mtDNA evolution in amphibians. These results highlight the need for further investigation of the effects of life history on mutation rates. Current address (Andrew J. Crawford): Smithsonian Tropical Research Institute, Apartado 2072, Balboa, Ancon, Republic of Panama     

Cell Tropism Predicts Long-term Nucleotide Substitution Rates of Mammalian RNA Viruses

The high rates of RNA virus evolution are generally attributed to replication with error-prone RNA-dependent RNA polymerases. However, these long-term nucleotide substitution rates span three orders of magnitude and do not correlate well with mutation rates or selection pressures. This substitution rate variation may be explained by differences in virus ecology or intrinsic genomic properties. We generated nucleotide substitution rate estimates for mammalian RNA viruses and compiled comparable published rates, yielding a dataset of 118 substitution rates of structural genes from 51 different species, as well as 40 rates of non-structural genes from 28 species. Through ANCOVA analyses, we evaluated the relationships between these rates and four ecological factors: target cell, transmission route, host range, infection duration; and three genomic properties: genome length, genome sense, genome segmentation. Of these seven factors, we found target cells to be the only significant predictors of viral substitution rates, with tropisms for epithelial cells or neurons (P<0.0001) as the most significant predictors. Further, one-tailed t-tests showed that viruses primarily infecting epithelial cells evolve significantly faster than neurotropic viruses (P<0.0001 and P<0.001 for the structural genes and non-structural genes, respectively). These results provide strong evidence that the fastest evolving mammalian RNA viruses infect cells with the highest turnover rates: the highly proliferative epithelial cells. Estimated viral generation times suggest that epithelial-infecting viruses replicate more quickly than viruses with different cell tropisms. Our results indicate that cell tropism is a key factor in viral evolvability.     

百合目叶绿体rbcL基因核苷酸替代速率检测

分析了百合目主要类群叶绿体中编码核酮糖1,5二磷酸羧化氧化酶大亚基rbcL基因的42条序列,使用RRTree相对速率检测方法,详细研究rbcL基因在百合目7科间同义替代速率和非同义替代速率的变化.相对速率检测显示:百合目内秋水仙科(Colchicaceae)的同义替代速率和非同义替代速率均最快,金梅草科(Campynemat-aceae)同义替代速率最慢,百合科(Liliaceae)的非同义替代速率最慢,但在百合目各科间,无论同义替代速率还是非同义替代速率差异均不显著.     

Intrageneric Phylogeny ofAcomys(Rodentia, Muridae) Using Mitochondrial Gene Cytochromeb

This paper investigates interspecies relationships within the genusAcomys(spiny mice) by analyzing entire mitochondrial cytochromebgene (1141 bp). This gene provides strong phylogenetic signal, as shown by high support of the topology obtained (bootstrap value and RNA support number). The phylogeny is congruent with inferences from allozymes for the species considered. Controversial taxonomy ofAcomys cahirinus, dimidiatus, airensis,andignitusis clarified, with their specific ranks confirmed on the basis of tree topology and nucleotide distances. Phylogenetic relationship between the undescribed speciesAcomyssp. from west Africa andA. airensisargue in favor of two distinct colonization events in this zone.     

Comparing Patterns of Nucleotide Substitution Rates among Chloroplast Loci Using the Relative Ratio Test

Even when several genetic loci are used in molecular evolutionary studies, each locus is typically analyzed independently of the others. This type of approach makes it difficult to study mechanisms and processes that affect multiple genes. In this work we develop a statistical approach for the joint analysis of two or more loci. The tests we propose examine whether or not nucleotide substitution rates across evolutionary lineages have the same relative proportions at two loci. Theses procedures are applied to 33 genes from the chloroplast genomes of rice, tobacco, pine, and liverwort. With the exception of five clearly distinct loci, we find that synonymous substitution rates tend to change proportionally across genes. We interpret these results to be consistent with a ``lineage effect' acting on the entire chloroplast genome. In contrast, nonsynonymous rates do not change proportionally across genes, suggesting that locus-specific evolutionary effects dominate patterns of nonsynonymous substitution.     

Synonymous Substitution Rates in Enterobacteria

It has been shown previously that the synonymous substitution rate between Escherichia coli and Salmonella typhimurium is lower in highly than in weakly expressed genes, and it has been suggested that this is due to stronger selection for translational efficiency in highly expressed genes as reflected in their greater codon usage bias. This hypothesis is tested here by comparing the substitution rate in codon families with different patterns of synonymous codon use. It is shown that the decline in the substitution rate across expression levels is as great for codon families that do not appear to be subject to selection for translational efficiency as for those that are. This implies that selection on translational efficiency is not responsible for the decline in the substitution rate across genes. It is argued that the most likely explanation for this decline is a decrease in the mutation rate. It is also shown that a simple evolutionary model in which synonymous codon use is determined by a balance between mutation, selection for an optimal codon, and genetic drift predicts that selection should have little effect on the substitution rate in the present case.     

Rates of Nucleotide Substitution in Angiosperm Mitochondrial DNA Sequences and Dates of Divergence Between Brassica and Other Angiosperm Lineages

We obtained 16 nucleotide sequences (∼1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16–0.23 × 10⁻⁹ substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5–0.7 × 10⁻⁹ substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170–235 million years (Myr) for the monocot–dicot split, 112–156 Myr for the Brassicaceae–Lettuce split, 14.5–20.4 Myr for the Brassica–Arabidopsis split, and 14.5–20.4 Myr for the Arabidopsis–Arabideae split. Received: 14 July 1998 / Accepted: 1 October 1998     

雄全异株植物白牛槭功能性状与碳素含量关联性研究

以雄全异株植物白牛槭为研究对象,在3个不同的花果发育期(盛花期、果实膨大期和果实成熟期)将雄全异株繁育系统和植物功能性状相结合,从功能性状与碳元素关联性角度研究植物繁殖和营养生长过程,探讨白牛槭适应环境的策略以及其两性植株在营养与繁殖之间的权衡关系。结果显示:(1)白牛槭枝叶功能性状在同一花果发育期内,除果实膨大期两性植株与雄株的叶干物质含量有显著差异外,其他2个花果发育期两性植株与雄株比叶面积(SLA)、叶干物质含量(LDMC)和枝干物质含量(BDMC)均无显著差异。不同花果发育期(盛花期、果实膨大期和成熟期)之间枝叶功能性状差异极显著,两性植株盛花期与果实膨大期的LDMC差异显著,果实膨大期和成熟期的SLA差异显著,而雄株并不存在这样的差异。(2)两性植株LDMC与BDMC具有极显著正相关关系,而雄株LDMC与BDMC之间呈显著相关关系,较两性植株弱。(3)同一花果发育期内,两性植株与雄株的叶碳含量、枝碳含量差异不显著;枝碳含量在不同花果发育期间差异极显著,大小顺序为:盛花期果实膨大期果实成熟期。(4)两性植株的枝叶碳含量与枝叶干物质含量均呈极显著正相关关系,雄树仅叶碳含量与枝干物质含量呈显著正相关关系。研究表明,白牛槭两性植株与雄株的繁殖和营养生长活动具有明显阶段性,两性植株在资源获取及养分保持能力方面显著大于雄株;在叶片与枝条之间联系紧密程度方面亦是如此,说明两性植株的养分制造及传输系统具有高度的一致性,这是两性植株面临着繁殖压力而做出的适应环境的策略之一,并且两性植株并没有因此而降低其营养生长,这可能与两性植株自身存在制造养分的补偿机制有关。     

Nucleotide metabolism variability in Drosophila melanogaster

Summary We have studied the metabolic variability within different wild-type strains of Drosophila melanogaster for resistance to antimetabolites (aminopterin, 8-azaguanine), the target enzymatic activities (dihydrofolate reductase, hypoxanthine guanine phosphoribosyltransferase) and capacity to survive on minimal medium with or without exogenous bases or nucleosides (thymidine, hypoxanthine). No correlation was found between dihydrofolate reductase activity and resistance to aminopterin. The results indicated the importance of salvage pathways in the resistance mechanisms in Drosophila.     

木聚糖酶分子的结构区域

多数木聚糖酶分子中含有多个功能区域,这些区域包括催化区、纤维素结合区、木聚糖结合区、连接序列、重复序列、热稳定区、Ｃｅｌｕｌｏｓｏｍｅ－Ｄｏｃｋｉｎｇ区及其它未知功能的非催化区,它们担负着不同的生物功能,不同来源的木聚糖酶分子的功能区域之间同源性或高或低。     

Estimating the Variability of Substitution Rates

Suppose that amino acid or nucleotide data are available for a homologous gene in several species which diverged from a common ancestor at about the same time and that substitution rates between all pairs of species are calculated, correcting as necessary for multiple substitutions and for back and parallel substitutions. The variances and covariances of these corrected substitution rates are evaluated, and are used to construct a new test for uniformity (constancy of the molecular clock) and to find the best estimates of substitution rates in individual lineages with their standard errors. A substantial bias may arise if the effect of correcting the pairwise substitution rates is ignored.     

云南重楼ITS序列单核苷酸多态性与甾体皂苷特征的相关性分析

为了解云南重楼(Paris polyphylla var. yunnanensis) ITS序列的单核苷酸多态性(SNP)特征与其甾体皂苷构成特征的相关性,并探讨其对药材质量稳定性的影响,利用MegAlign软件对37份云南重楼样本的ITS序列进行对比,根据SNP位点进行分型;采用HPLC法测定7种甾体皂苷成分(重楼皂苷Ⅰ、Ⅱ、Ⅴ、Ⅵ、Ⅶ、H和薯蓣皂苷);用SPSS 25.0软件和SIMCA-P 15.0软件对各基因型甾体皂苷构成特征进行统计分析。结果表明,云南重楼ITS序列存在40个双等位多态性位点,其中碱基转换32个,碱基颠换8个,根据SNP特征可划分为2类基因型YN-I和YN-II。6种甾体皂苷(重楼皂苷Ⅰ、Ⅱ、Ⅵ、Ⅶ、H、薯蓣皂苷)在云南重楼广泛分布,而重楼皂苷Ⅴ稀少。YN-I和YN-II的药典指标成分总含量分别为1.070%和0.93%,样本合格率分别为68.42%和77.78%;YN-I的甾体皂苷总含量为1.65%,YN-II为1.32%。方差分析表明,2类基因型的甾体皂苷特征存在一定程度的区别,但药典指标成分无显著差异,药材应该具有等效性。聚类分析和主成分分析表明,YN-Ⅰ的离散度更高,YN-Ⅱ的聚集度更好,表明YN-II的药材质量更加稳定,植株个体的甾体皂苷合成和累积差异更小,YN-II的SNP特征对云南重楼良种选育的分子遗传标记筛选具有重要意义。     

Estimating Substitution Rates in Ribosomal RNA Genes

A model is introduced describing nucleotide substitution in ribosomal RNA (rRNA) genes. In this model, substitution in the stem and loop regions of rRNA is modeled with 16- and four-state continuous time Markov chains, respectively. The mean substitution rates at nucleotide sites are assumed to follow gamma distributions that are different for the two types of regions. The simplest formulation of the model allows for explicit expressions for transition probabilities of the Markov processes to be found. These expressions were used to analyze several 16S-like rRNA genes from higher eukaryotes with the maximum likelihood method. Although the observed proportion of invariable sites was only slightly higher in the stem regions, the estimated average substitution rates in the stem regions were almost two times as high as in the loop regions. Therefore, the degree of site heterogeneity of substitution rates in the stem regions seems to be higher than in the loop regions of animal 16S-like rRNAs due to presence of a few rapidly evolving sites. The model appears to be helpful in understanding the regularities of nucleotide substitution in rRNAs and probably minimizing errors in recovering phylogeny for distantly related taxa from these genes.     

Nucleotide Substitution Rate of Mammalian Mitochondrial Genomes

We present here for the first time a comprehensive study based on the analysis of closely related organisms to provide an accurate determination of the nucleotide substitution rate in mammalian mitochondrial genomes. This study examines the evolutionary pattern of the different functional mtDNA regions as accurately as possible on the grounds of available data, revealing some important ``genomic laws.' The main conclusions can be summarized as follows. (1) High intragenomic variability in the evolutionary dynamic of mtDNA was found. The substitution rate is strongly dependent on the region considered, and slow- and fast-evolving regions can be identified. Nonsynonymous sites, the D-loop central domain, and tRNA and rRNA genes evolve much more slowly than synonymous sites and the two peripheral D-loop region domains. The synonymous rate is fairly uniform over the genome, whereas the rate of nonsynonymous sites depends on functional constraints and therefore differs considerably between genes. (2) The commonly accepted statement that mtDNA evolves more rapidly than nuclear DNA is valid only for some regions, thus it should be referred to specific mitochondrial components. In particular, nonsynonymous sites show comparable rates in mitochondrial and nuclear genes; synonymous sites and small rRNA evolve about 20 times more rapidly and tRNAs about 100 times more rapidly in mitochondria than in their nuclear counterpart. (3) A species-specific evolution is particularly evident in the D-loop region. As the divergence times of the organism pairs under consideration are known with sufficient accuracy, absolute nucleotide substitution rates are also provided. Received: 11 May 1998 / Accepted: 2 September 1998     

Dynamically Driven Ligand Selectivity in Cyclic Nucleotide Binding Domains

One of the mechanisms that minimize the aberrant cross-talk between cAMP- and cGMP-dependent signaling pathways relies on the selectivity of cAMP binding domains (CBDs). For instance, the CBDs of two critical eukaryotic cAMP receptors, i.e. protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), are both selectively activated by cAMP. However, the mechanisms underlying their cAMP versus cGMP selectivity are quite distinct. In PKA this selectivity is controlled mainly at the level of ligand affinity, whereas in EPAC it is mostly determined at the level of allostery. Currently, the molecular basis for these different selectivity mechanisms is not fully understood. We have therefore comparatively analyzed by NMR the cGMP-bound states of the essential CBDs of PKA and EPAC, revealing key differences between them. Specifically, cGMP binds PKA preserving the same syn base orientation as cAMP at the price of local steric clashes, which lead to a reduced affinity for cGMP. Unlike PKA, cGMP is recognized by EPAC in an anti conformation and generates several short and long range perturbations. Although these effects do not alter significantly the structure of the EPAC CBD investigated, remarkable differences in dynamics between the cAMP- and cGMP-bound states are detected for the ionic latch region. These observations suggest that one of the determinants of cGMP antagonism in EPAC is the modulation of the entropic control of inhibitory interactions and illustrate the pivotal role of allostery in determining signaling selectivity as a function of dynamic changes, even in the absence of significant affinity variations.In eukaryotes, protein kinase A (PKA)² and the exchange protein directly activated by cAMP (EPAC) are two major receptors for the cAMP second messenger (1–4). The activities of both PKA and EPAC are modulated in a cAMP-dependent manner through cAMP binding domains (CBDs) (1–4). In all isoforms of PKA, two tandem CBDs, denoted as CBD-A and CBD-B, are part of the regulatory subunit (R), in which they are preceded by an N-terminal dimerization docking module and a linker region (Fig. 1a) (1, 3). In the inactive state PKA exists as a tetrameric holo-enzyme complex, including two regulatory (R) subunits and two catalytic (C) subunits (1, 3). Binding of cAMP to the CBDs of the R subunits results in the release of the C subunits and in the activation of the kinase function (1, 3).Open in a separate windowFIGURE 1.Schematic representation of the domain organization in the regulatory subunit I-α of PKA (a) and in EPAC (b).The black circles indicate cAMP. a, D/D is the dimerization docking domain; the inhibitory site is shown in orange, and the two tandem cAMP binding domains, CBD-A and CBD-B, are highlighted in different shades of green. b, DEP, disheveled-egl-10-pleckstrin domain; REM, Ras exchange motif; RA, Ras-associated module, and the CDC25HD catalytic domain are represented in gray, green, orange, blue, and yellow, respectively. The black dashed line and the empty cAMP circle in EPAC2 indicate that this domain is not strictly necessary for the cAMP-dependent GEF activity. The module with question mark in EPAC1 denotes an unknown function for this domain. a and b, the CBD in light green shown below the full-length protein represents the construct used for the NMR studies. c, sequence alignment of the CBDs of bovine RIα- domain A and human EPAC1. Fully conserved residues are highlighted in green; cyan denotes conservation for the functional group only, and yellow indicates the residues present only in one of the CBDs. The secondary structure of apo-EPAC2_m (PDB ID 1O7F) is shown in red.Unlike PKA, EPAC is a single-chain protein that functions as a guanine nucleotide-exchange factor (GEF) for the small GTPase Rap1 and Rap2 (2, 4). The domain organization of EPAC includes an N-terminal regulatory region (RR) and a C-terminal catalytic region (CR) (Fig. 1b). There are two known homologous isoforms of EPAC, i.e. EPAC1 and EPAC2. One of the key differences between EPAC1 and EPAC2 is that in the former there is only a single CBD, whereas in the latter there are two noncontiguous CBDs, i.e. CBD-A and CBD-B. However, CBD-A has been shown not to be strictly necessary for the cAMP-dependent activation of EPAC (2, 4).Both PKA and EPAC are critical for the regulation of a wide range of cAMP-dependent physiological processes (1–4), and impaired activity of these cAMP sensors has been implicated in cardiovascular pathology, diabetes, and Alzheimer disease (1–4). Therefore, PKA and EPAC represent attractive therapeutic targets. However, the design and development of specific drug leads targeting the CBDs of either of these two eukaryotic protein systems require an in depth analysis of how PKA and EPAC selectively recognize and allosterically respond to diverse cNMPs. For instance, the cAMP and cGMP second messengers control distinct groups of essential signaling pathways (1–6). It is therefore critical to minimize the cross-talk between the cAMP- and cGMP-dependent cellular responses. Although in vivo the selective control of the cAMP- and/or cGMP-dependent signaling pathways is a complex process that depends on multiple factors, including the modulation of cNMP synthesis, degradation, and compartmentalization (5), one of the key mechanisms to reduce the cAMP/cGMP cross-talk relies on the ability of both PKA and EPAC CBDs to sense selectively cAMP as opposed to cGMP.Despite the fact that both PKA and EPAC CBDs are cAMP-selective sensors, these two signaling systems adopt different mechanisms to implement their cAMP-selective response. Specifically, in the PKA system cGMP is an agonist of cAMP, i.e. cGMP is able to activate PKA once it binds the R subunit. However, the affinity of cGMP for the PKA R subunit is significantly lower than that of cAMP, resulting in an activation constant that is 2 orders of magnitude higher than that of cAMP (i.e. K_a of 21 ± 2 nm for cAMP and of 4100 ± 20 nm for cGMP) (7, 8). Unlike PKA, EPAC preserves approximate micromolar affinities for both cAMP and cGMP, but in EPAC the latter cNMP is an antagonist of cAMP (9, 10), i.e. cGMP, like other N⁶-substituted cAMP analogs, binds effectively to the CBD of EPAC but fails to fully activate its GEF activity (9, 10).The molecular basis for the cGMP antagonism selectively observed in EPAC but not in PKA is currently not fully understood. An initial hypothesis to explain the antagonist function of cGMP in EPAC has been recently proposed based on the structure of the ternary (S_p)-cAMPS-EPAC2_m-Rap1 complex (11), which shows that the N⁶ of the (S_p)-cAMPS agonist forms an hydrogen-bond with the backbone carbonyl oxygen of Lys-450 located in the lid region (supplemental Table S1) (11). The disruption of this hydrogen bond by cGMP has been hypothesized to result in the inhibition of EPAC activation (11). However, a similar backbone hydrogen bond between the N⁶ of cAMP and the backbone carbonyl oxygen of Arg-632 has been observed also in the CBDs of the hyperpolarization-activated cyclic nucleotide-modulated channels (HCN) (supplemental Table S1), for which cGMP, like in PKA, is not an antagonist (12). This observation suggests that the elimination of the cAMP N⁶ hydrogen bond alone may not be sufficient to fully explain why cGMP is a cAMP antagonist with respect to the activation of EPAC. Furthermore, the previously proposed hypothesis based on the simple disruption by cGMP of the N⁶ hydrogen bond does not consider the possibility that cGMP may adopt an EPAC-bound conformation different from that of cAMP and/or that cGMP may affect also the inhibitory interactions between the ionic latch residues of the EPAC RR and the CDC25HD catalytic domain. These RR/CR salt bridges stabilize the EPAC system in an overall “closed” topology, whereby the RR sterically occludes access of Rap1 into the catalytic domain of EPAC (13). cAMP binding results in increased picosecond to nanosecond and millisecond to microsecond dynamics at the ionic latch region, which in turn leads to an increased entropic penalty for the inhibitory interactions mediated by the ionic latch (14). In other words, cAMP is able to weaken the inhibitory interactions between the regulatory and catalytic regions of EPAC by increasing the entropic cost associated with the formation of the cluster of ionic latch salt bridges between these two functional segments. This dynamically driven mechanism contributes to the observed cAMP-dependent shift toward active “open” conformations of EPAC, and we hypothesize that one of the effects of cGMP is to perturb the dynamic patterns of the EPAC CBD, thus altering the entropic control of the inhibitory interactions.To test our hypotheses on cGMP agonism/antagonism and to further understand the molecular mechanisms underlying the different signaling responses of PKA and EPAC to the two endogenous second messengers cAMP and cGMP, here we present a comparative NMR analysis of cGMP binding and allostery for the critical CBDs of both PKA and EPAC. These results were also compared with the data on the same domains in their apo- and cAMP-bound states (14–20). All these studies rely on the RIα-(119–244) and the related EPAC1_h-(149–318) constructs (Fig. 1), which have been previously validated as models of the essential CBDs of PKA and EPAC, respectively (14–20).Our comparative NMR analysis has revealed that the structure, dynamics, and allosteric activation pathways of the PKA CBD-A are not significantly altered when cAMP is replaced by cGMP. However, significant differences between these two cNMPs are found for the EPAC1_h CBD at the level of both ligand recognition and modulation of dynamic modes, leading to a mechanism in which cGMP shifts the activation equilibrium of EPAC toward the auto-inhibited state, thus accounting for its antagonistic function.     

Evolution of a Mitochondrial CytochromebGene Sequence in the Species-Rich GenusSebastes(Teleostei, Scorpaenidae) and Its Utility in Testing the Monophyly of the SubgenusSebastomus

The ecologically diverse and species-rich rockfishes (Sebastes) are a useful group for the study of marine speciation. The monophyly of the genus is generally accepted, as is the validity of most of the numerous species found along the West Coast of North America. However, the subgeneric groupings that would help in the proposal and interpretation of various speciation schemes are poorly supported based on widely overlapping morphological characters. The use of genetic characters provides an alternative approach. In this study, we present the first quantitative analysis supporting the monophyly of all 14 species of the subgenusSebastomusamong 54 congeners. The structural features and evolution of the 750 bp of the cytochromebgene used to test the monophyly were similar to those of other vertebrates. Low levels of intrageneric sequence divergence (<10%) and incipient levels of saturation at third-codon positions were found in the gene. The monophyly ofSebastomuswas supported in extensive phylogenetic analyses using distance, cladistic, and likelihood methods. Further corroboration was obtained from permutation- and simulation-based statistical tests.     

谷氏菌素生物合成基因gouC和gouD的功能研究北大核心CSCD

【目的】由于谷氏菌素产生菌禾粟链霉菌的遗传操作效率较低,本研究在谷氏菌素生物合成基因簇成功异源表达的基础上,通过在异源宿主天蓝色链霉菌M1146中阻断谷氏菌素生物合成基因gouC和gouD,研究其在谷氏菌素生物合成中的作用,为谷氏菌素生物合成途径的阐明奠定基础。【方法】以含有谷氏菌素生物合成基因簇的黏粒D6-4H为基础,通过PCR-targeting的方法分别将gouC和gouD敲除得到重组质粒p GOUe-ΔC和p GOUe-ΔD。通过接合转移将这两个重组质粒分别导入天蓝色链霉菌M1146中,获得gouC和gouD的缺失突变株M1146-GOUe-ΔC和M1146-GOUe-ΔD,通过HPLC分析突变株的谷氏菌素中间产物积累情况,分离纯化后对其进行结构鉴定和生物活性检测。【结果】gouC和gouD的缺失均导致谷氏菌素不能合成,突变株发酵液中积累了不同的中间产物,生物活性分析发现这些中间产物均失去了对肿瘤细胞的抑制活性。【结论】gouC和gouD是谷氏菌素生物合成的重要基因,与谷氏菌素肽基部分的肌氨酸残基合成相关。本研究为阐明谷氏菌素的生物合成机制提供了更多的依据。     

蕙兰CfCIN基因克隆及其功能分析

以蕙兰(Cymbidium faberi Rolfe)为试验材料,采用RT-PCR技术克隆了TCP家族的CIN同源基因,开放阅读框长1 161bp,编码386个氨基酸,将其命名为CfCIN(GenBank登录号为KJ956809)。为进一步分析CfCIN的功能,构建了植物表达载体,采用农杆菌介导法转化非洲紫罗兰叶片,获得了转化植株并对转基因植株进行了性状分析。结果显示:与野生型非洲紫罗兰叶片相比,转基因植株的叶片更大,由圆形变为卵圆形,叶缘由平整光滑变为有缺刻且稍向后卷曲,叶脉明显,叶柄红,花器官形状变化不明显。研究表明,CfCIN可能参与调控植物叶片的形态建成。     

Variation in the Pattern of Nucleotide Substitution Across Sites

A model of nucleotide substitution that allows the transition/transversion rate bias to vary across sites was constructed. We examined the fit of this model using likelihood-ratio tests by analyzing 13 protein coding genes and 1 pseudogene. Likelihood-ratio testing indicated that a model that allows variation in the transition/transversion rate bias across sites provided a significant improvement in fit for most protein coding genes but not for the pseudogene. When the analysis was repeated with parameters estimated separately for first, second, and third codon positions, strong heterogeneity was uncovered for the first and second codon positions; the variation in the transition/transversion rate was generally weaker at the third codon position. The transition rate bias and branch lengths are underestimated when variation in the transition/transversion rate was not accommodated, suggesting that it may be important to accommodate variation in the pattern of nucleotide substitution for accurate estimation of evolutionary parameters. Received: 4 November 1997 / Accepted: 19 May 1998