β-KL离散量在识别DNA编码区域中的应用

根据DNA序列的‘终止密码子’及其‘逆补终止密码子’的分布情况,给出一种新的DNA序列向量的构建方法,运用Shannon熵相关理论,对Jensen-Shannon离散量和KL离散量进行了修正和比较。试验表明,该方法在预测DNA序列的基因编码与非编码区边界的效率上是86%显著高于Bernaola等人提出的70%。     

基于统计CPG岛的基因预测方法

随着以功能基因组学和蛋白质组学为主要研究内容的后基因组时代的来临,人们面对着生物信息的数据呈指数增长,如何通过有效的计算方法由核酸和蛋白质的序列推导出它们的结构和功能,特别是识别DNA序列中编码蛋白质的基因预测问题是迫切需要解决的研究课题之一.本文在CpG岛对研究基因编码的特殊生物意义下,通过三种方法确定CpG岛的位置,并在此基础上,结合一种新的DNA序列字母向量,利用信息熵离散量预测基因序列,提高了识别基因编码的效率,而且计算的时间有显著的减少.     

藏羚羊PGC-1α基因编码区的克隆与分析

以藏羚羊(Pantholops hodgsonii)及同海拔分布的藏系绵羊(Tibetan Sheep)的心肌组织为材料,提取总RNA,利用逆转录聚合酶链反应(RT-PCR)技术扩增出过氧化物酶体增生物激活受体γ辅激活因子-1α(PGC-1α)的基因编码区cDNA片段,与载体连接构建重组质粒,经转化、扩增培养、鉴定后测序。利用生物信息学方法分析显示,藏羚羊和藏系绵羊的PGC-1α基因编码区长度均为2 349 bp,编码797个氨基酸(GenBank登录号分别为:JF449959和JF449960);与其他脊椎动物PGC-1α基因的核苷酸及氨基酸序列相似性达到90%以上;其包含RNA/DNA结合位点、RNA识别基序(RRM)、与核呼吸因子1(NRF-1)及肌细胞增强因子2C(MEF2C)相互作用的区域、富含丝氨酸/精氨酸的结构域、负调节功能结构域、LXXLL模体以及TPPTTPP和DHDYCQ两个保守序列,14个氨基酸差异性位点位于以上部分功能结构域中;此外,磷酸化位点的预测提示藏羚羊可能存在一个潜在的蛋白激酶G的磷酸化位点(第329位的苏氨酸)。本研究成功克隆出了藏羚羊PGC-1α基因的编码区序列,为从能量代谢角度深入探讨藏羚羊适应高原的分子生物学机制提供了新的思路。     

人α－肿瘤坏死因子基因的高效表达

用人工合成的α－肿瘤坏死因子(TNF—α)基因构建了五个不同的表达质粒,它们不同之处是SD序列与起始密码子ATG问距离(D)各异．计算机模拟计算出翻译起始区域(TIR)中二级结构的最小生成自由能．以(D)为6个核苷酸时最小(绝对值)。它的表达效率也最高,产物TNF—α可达菌体总蛋白的60％．密码子的选用对表达效率有很大的影响,故人工合成TNF－α基因(选用大肠杆菌偏爱的密码子)的表达效率高于sc－DNA(对部分密码子改造的半合成eDNA)．     

重组人降钙素基因相关肽融合表达及纯化初步研究

采用大肠杆菌偏爱的密码子人工合成hαCGRP基因,构建了原核融合表达载体,对融合蛋白成功地进行了表达、纯化,Western免疫印迹验证该蛋白具有αCGRP抗原性,为下一步hαCGRP纯品的获得及动物实验的研究奠定基础。     

人类蛋白编码基因局部GC水平相关性分析

GC含量是基因组DNA序列碱基组成的重要特征, 蕴涵基因结构、功能和进化信息。文中通过从公共数据库提取7 992个非冗余的人类蛋白质编码基因DNA序列, 分析了基因序列不同区域的局部GC含量和相关性。结果表明: 基因局部GC含量呈现不均一性, 5′非翻译区GC水平最高, 为62.56%; 而3′非翻译区GC水平最低, 为43.97%。3′侧翼序列的GC含量能较好地代表基因所在区域DNA长片段的GC水平。虽然开放阅读框的GC含量比内含子、3′非翻译区和3′侧翼序列的GC含量高, 但4个区域的GC含量之间均存在较高的相关性。密码子第三位置的平均GC含量(GC3)为58.09%, 显著高于密码子第一位置和第二位置的GC含量, 且与开放阅读框的GC水平高度相关, 相关系数高达0.91。GC3与内含子、3′非翻译区、3′侧翼序列的GC水平相关性也较高, GC3对3′侧翼序列的GC含量的直线回归斜率为1.25。因此, GC3可作为基因所在区域GC水平变化的敏感性指标。而密码子第一位置和第二位置以及5′侧翼序列和5′非翻译区GC水平与基因其他区域的GC水平的相关性较弱。该研究结果提示: 基因蛋白编码区密码子第三位置、内含子、3′非翻译区和3′侧翼序列的碱基可能经历了相近的进化过程, 而蛋白编码区密码子第一位置和第二位置、5′侧翼序列和5′非翻译区由于功能的需要而经历了不同的突变和选择。     

基于支持向量机的人类5’非翻译区剪接位点识别

基因非编码区域剪接位点的识别是基因识别中一个非常具有挑战性的问题,尤其是5’非翻译区中剪接位点的识别。与一般剪接位点不同,5’非翻译区剪接位点的两侧不存在由编码到非编码的状态转移,所以通常的剪接位点识别算法在非翻译区的性能不太理想。文章采用了基于支持向量机的方法对5’非翻译区中的剪接位点进行识别。为了提高识别精度,采用了基于矩阵相似性度量的核函数参数选取方法,它能够简单快速地确定合适的核函数参数,进而提高核函数的识别性能。通过实验验证,经过参数选择后的支持向量机能够较好地识别5＇非翻译区剪接位点。     

白鹅催乳素基因的克隆及诱导表达条件的优化

摘要: 运用RT-PCR方法, 从白鹅脑垂体总RNA中扩增得到了催乳素(Prolactin, PRL)基因编码区序列cDNA, 并将其克隆到pMD18-T载体上。DNA序列分析表明, PRL cDNA包括终止密码子在内的长度为690 bp,编码230个氨基酸残基的蛋白质, 与皖西白鹅的有所差异, 二者碱基同源性在99.57%, 氨基酸同源性达99.56%。将PRL基因编码区序列cDNA定向克隆到表达载体pET-32a (+)中, 构建表达质粒pET-32a(+)-PRL。该质粒的BL21 (DE3)转化菌在IPTG的诱导下可表达PRL基因融合蛋白, IPTG终浓度1 mmol/L, 37℃, 诱导4 h表达量最高, 表达量约占菌体总蛋白的28.96%。     

鲁西黄牛α干扰素基因的克隆及表达

提取黄牛血液基因组DNA,PCR扩增α干扰素基因,重组到pET32a 表达载体中。测序结果表明,扩增片段含有498bp的ORF,可编码166个氨基酸的成熟蛋白,与已报道的牛α干扰素C亚型氨基酸组成同源性为97.6%。构建原核表达载体pET32a /BoIFN-α,SDS-PAGE分析蛋白质表达水平,IPTG诱导后表达的融合蛋白分子量为40ku,表达量占菌体总蛋白的26.7%。结果从鲁西黄牛中克隆了IFN-α基因的一种新亚型,即BoIFN-αC2,构建原核表达质粒,并实现了高效表达,为重组牛干扰素的开发奠定了基础。     

合成编码蛋白质基因的设计

 本文以牛生长激素基因为例,对合成编码蛋白质基因的微机设计原理进行了探讨。微机程序的编制采用高级BASIC语言,在IBM-PC微机上完成。设计合成编码蛋白质基因的要点为:(1)按照宿主系统中高表达蛋白质基因对密码子的使用频率选用氨基酸密码子,以期合成基因得到高效表达;(2)对较大的合成基因设计有能够进行分段克隆的酶切位点,从而将一个大的基因分解成为多个基因片段的合成,减少了酶促连接化学合成DNA片段的步骤;(3)对于酶促连接化学合成DNA片段有干扰的重复顺序和互补顺序,则利用变换简并密码子的办法予以消除。     

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.     

Measuring the Coding Potential of Genomic Sequences Througha Combination of Triplet Occurrence Patterns and RNY Preference

The distribution of n-tuplet frequencies is shown to strongly correlate with functionality when examining a genomic sequence in a reading-frame specific manner. The approach described herein applies a coarse-graining procedure, which is able to reveal aspects of triplet usage that are related to protein coding, while at the same time remaining species independent, based on a simple summation of suitable triplet occurrences measures. These quantities are ratios of simple frequencies to suitable mononucleotide-frequency products promoting the incidence of the RNY motif, preferred in the most widely used codons. A significant distinction of coding and noncoding sequences is achieved.Reviewing Editor: Dr. Massimo Di Giulio     

Genomic divergence across ecological gradients in the Central African rainforest songbird (Andropadus virens)

The little greenbul, a common rainforest passerine from sub‐Saharan Africa, has been the subject of long‐term evolutionary studies to understand the mechanisms leading to rainforest speciation. Previous research found morphological and behavioural divergence across rainforest–savannah transition zones (ecotones), and a pattern of divergence with gene flow suggesting divergent natural selection has contributed to adaptive divergence and ecotones could be important areas for rainforests speciation. Recent advances in genomics and environmental modelling make it possible to examine patterns of genetic divergence in a more comprehensive fashion. To assess the extent to which natural selection may drive patterns of differentiation, here we investigate patterns of genomic differentiation among populations across environmental gradients and regions. We find compelling evidence that individuals form discrete genetic clusters corresponding to distinctive environmental characteristics and habitat types. Pairwise F_ST between populations in different habitats is significantly higher than within habitats, and this differentiation is greater than what is expected from geographic distance alone. Moreover, we identified 140 SNPs that showed extreme differentiation among populations through a genomewide selection scan. These outliers were significantly enriched in exonic and coding regions, suggesting their functional importance. Environmental association analysis of SNP variation indicates that several environmental variables, including temperature and elevation, play important roles in driving the pattern of genomic diversification. Results lend important new genomic evidence for environmental gradients being important in population differentiation.     

An Evaluation of Measures of Synonymous Codon Usage Bias

Synonymous codons are not generally used at equal frequencies, and this trend is observed for most genes and organisms. Several methods have been proposed and used to estimate the degree of the nonrandom use of the different synonymous codons. The estimates obtained by these methods, however, show different levels of both precision and dispersion when coding regions of a finite number of codons are under analysis. Here, we present a study, based on computer simulation, of how the different methods proposed to evaluate the nonrandom use of synonymous codons are affected by the length of the coding region analyzed. The results show that some of these methods are heavily influenced by the number of codons and that the comparison of codon usage bias between coding regions of different lengths shows a methodological bias under different conditions of nonrandom use of synonymous codons. The study of the dispersion of the estimates obtained by the different methods gives, on the other hand, an indication of the methods to be applied to compare values of codon usage bias among coding regions of equivalent length. Received: 10 September 1997 / Accepted: 23 March 1998     

A universal mechanism generating clusters of differentiated loci during divergence‐with‐migration

Genome‐wide patterns of genetic divergence reveal mechanisms of adaptation under gene flow. Empirical data show that divergence is mostly concentrated in narrow genomic regions. This pattern may arise because differentiated loci protect nearby mutations from gene flow, but recent theory suggests this mechanism is insufficient to explain the emergence of concentrated differentiation during biologically realistic timescales. Critically, earlier theory neglects an inevitable consequence of genetic drift: stochastic loss of local genomic divergence. Here, we demonstrate that the rate of stochastic loss of weak local differentiation increases with recombination distance to a strongly diverged locus and, above a critical recombination distance, local loss is faster than local “gain” of new differentiation. Under high migration and weak selection, this critical recombination distance is much smaller than the total recombination distance of the genomic region under selection. Consequently, divergence between populations increases by net gain of new differentiation within the critical recombination distance, resulting in tightly linked clusters of divergence. The mechanism responsible is the balance between stochastic loss and gain of weak local differentiation, a mechanism acting universally throughout the genome. Our results will help to explain empirical observations and lead to novel predictions regarding changes in genomic architectures during adaptive divergence.     

Construction of a multiframe vector to express coding sequences in Escherichia coli

Cloning of foreign DNA fragments for coding sequence analysis in Escherichia coli usually involves sets of three vectors. To simplify this, we constructed an expression vector named pMFV7 containing three ATG codons in different frames downstream of a Shine-Dalgarno sequence, assuming that the ribosome can use any of the three start codons in an alternative manner. Translation beginning at either of the start codons would drive the expression of any coding fragment cloned downstream. To test the feasibility of this proposal, we cloned DNA fragments of the lacZ gene in each of the possible reading frames downstream from pMFV7 start codons. Sequence analysis of the N-terminus regions around the fusion sites indicates that ribosomes indeed initiate translation at each of the three initiation codons. In one case, levels of beta-galactosidase activity depended largely on the N-terminus of the translation products. We conclude that pMFV7 may be useful for expressing coding sequences regardless of their reading frame.     

Computational prediction of MHC II-antigen binding supports divergent allele advantage and explains trans-species polymorphism

The major histocompatibility complex (MHC), coding for antigen presenting molecules of the adaptive immune system, represents one of the most polymorphic regions in the vertebrate genome. The exceptional polymorphism, which is potentially maintained by balancing selection under host-parasite coevolution, comprises excessive sequence divergence among alleles as well as ancient allelic lineages that predate species divergence (trans-species polymorphism). Here, the mechanisms that are proposed to maintain such sequence divergence and ancient lineages are investigated. Established computational antigen-binding prediction algorithms, which are based on empirical databases, are employed to determine the overlap in bound antigens among individual MHC class IIB alleles. The results show that genetically more divergent allele pairs experience less overlap and thus present a broader range of potential antigens. These findings support the divergent allele advantage hypothesis and furthermore suggest an evolutionary advantage explaining the maintenance of divergent allelic lineages, that is, trans-species polymorphism. In addressing a quantitative rather than qualitative aspect of MHC alleles, these insights highlight a new direction for future research on MHC evolution.     

One-Step Lipophilization of Proteins Using Fatty Acyl Anhydride

Nucleotide sequences of mRNAs were compared between major calcium-sensitive caseins of cow (α_s1-casein) and rat (α-casein). A best fit alignment of the two sequences showed homology of 81% and 69% for the 5′- and 3′-untranslated regions, respectively. Homology in the comparable coding region of the mature asl-casein (76% of total codons) was remarkably lower at amino acid level (46%) than at nucleotide level (69%). The low conservation at amino acid level is explained by the unusual nucleotide substitution pattern (random at all three positions of codons) in contrast to synonymous substitutions at the third position revealed on comparison of other related proteins. The evolutionary distances among the number of the casein family were estimated by comparing known nucleotide sequences of the signal peptides which were the most conserved coding regions in the family. The divergence time for most distantly related caseins (both rat α-casein/rat β-casein and rat α-casein/mouse ε-casein) was estimated to be about 170 million years.     

The extent of parental genotypic divergence determines maximal heterosis by increasing fertility in inter-subspecific hybrids of rice (Oryza sativa L.)

The magnitude of heterosis in F₁ hybrids is related not only to the performance of parents per se but also to the genetic diversity between two parents. The extent of genotypic divergence between hybrid rice parents was investigated at the molecular level, using two subsets of rice materials: a subset of doubled haploid (DH) lines derived from an Indica × Japonica cross (Gui630/02428) and another subset of Indica or Japonica lines representative of a broad spectrum of the Asian cultivated rice gene pool, including landraces, primitive cultivars, historically important cultivars, modern elite cultivars, super rice and parents of superior hybrids. 57 entries deliberately selected from the 81-DH lines (in total) were testcrossed to two widely used rice lines in China, photoperiod-sensitive genic male sterile (PGMS) N422s and thermo-sensitive genic male sterile (TGMS) Peiai64s. Results of the two sets of test-cross F₁ populations showed congruently that parental genotypic divergence has a relatively low impact on heterosis for the two yield components, i.e., panicle number and 1000-grain weight, but it has a great bearing on fertility parameters, i.e., filled grains per plant and seedset. Heterosis for grain yield in the two test-cross populations exhibited a sharp maximum when the proportion of Japonica alleles in the male parent was between 50 and 60%, so was the heterosis for fertility parameters correspondingly. Thus fertility parameters were the most sensitive and important factors which were influenced by the extent of parental genotypic divergence. Moreover, our results showed that parents with moderate extent of genotypic divergence played an important role in the use of inter-subspecific rice heterosis.