杜仲基因密码子使用模式分析

为了解杜仲基因密码子使用模式,该文以杜仲基因组密码子为研究对象,运用CodonW软件对杜仲的320个蛋白编码基因进行同义密码子相对使用频率(RSCU)分析、ENC-GC3s关联分析编码基因的密码子ENC值、PR2-plot偏倚分析编码基因的密码子碱基使用频率,并运用CUSP软件与Codon Usage Database软件对杜仲基因密码子的GC含量、使用频率与代表性物种烟草、拟南芥、大肠杆菌和酿酒酵母的密码子GC含量和使用频率进行比较。结果表明:杜仲基因密码子的RSCU>1的密码子有30个,其中18个以G/C结尾、12个以A/U结尾,说明杜仲基因密码子偏好以G/C结尾,且偏好性较强;有效密码子数(ENC)范围为30~60,该范围内的密码子距离标准曲线较远,其ENC值小,偏好性较强;PR2-plot偏倚分析碱基使用频率显示,G>C、U>A;杜仲与代表性物种的GC含量分析显示,杜仲的GC12、GC3以及平均GC含量均高于代表性物种;杜仲与代表性物种的密码子使用频率分析显示,杜仲与烟草、酿酒酵母的密码子偏好较为接近,杜仲与拟南芥、大肠杆菌的密码子偏好差距较大。杜仲是我国特有的珍贵中药材,对其进行密码子使用模式分析,并研究其密码子偏好规律,为杜仲植物基因工程中外源基因的改良及表达提供了理论基础。     

Cloning and sequencing of the HU-2 gene of Escherichia coli

Summary The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe. The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rhoindependent terminater site downstream of the termination codon (UAA) of the gene.     

槲蕨属叶绿体基因组密码子偏好性分析

叶绿体基因组密码子偏好性使用模式往往影响基因表达效率,为促进药用植物叶绿体基因工程的发展,提高槲蕨药用品质,该研究以川滇槲蕨、栎叶槲蕨和槲蕨三个近缘药用植物为材料,使用CodonW、CUSP和SPSS等软件分析其叶绿体基因组编码基因密码子使用偏好性,筛选出三个物种的最优密码子。结果表明:川滇槲蕨、栎叶槲蕨和槲蕨叶绿体基因组的有效密码子数(ENC)范围分别为40.10~61、40.33~61和40.15~61,其密码子偏好性较弱;ndhE、rpl22、rpl14、rpl20、ccsA、rps4和rpl16编码基因的ENC值差异较大,表明近缘物种中,部分基因的密码子偏好性存在一定差异;三个物种编码基因的ENC频数集中于-0.1~0.1之间,说明槲蕨属基因密码子偏好性主要受到突变的影响。川滇槲蕨12个最优密码子有6个和栎叶槲蕨相同,分别是UCU、ACU、GCU、CAA、AAA和GAU;栎叶槲蕨10个最优密码子有2个和槲蕨相同,分别是UUA和AUU,而川滇槲蕨与槲蕨无相同的最优密码子。该研究结果可为槲蕨属药用植物基因工程中外源基因的改良及其表达奠定基础。     

桃儿七叶绿体比较基因组学分析

为探究桃儿七（Sinopodophyllum hexandrum）不同叶绿体基因组特征,本研究以桃儿七5个叶绿体基因组为研究对象,借助生物信息学工具进行基因组图谱构建、重复序列分析、密码子偏好分析、反向重复序列区（inverted repeat,IR）/单拷贝区（single-copy,SC）边界分析、基因组序列比较分析及系统发育分析。结果表明：桃儿七5个叶绿体基因组全长为157 203–157 940 bp,为典型的叶绿体四分体结构,共注释出133–137个基因,说明桃儿七叶绿体基因组具有多样性。桃儿七不同叶绿体基因组简单重复序列（simple sequence repeat,SSR）位点不同,单核苷酸A/T占主要优势,散在重复序列包括正向重复、回文重复和反向重复3类。密码子偏好分析显示有效密码子（effective number of codon,ENc）值为51.14–51.17,密码子偏好性弱,GC与GC3s所占比例小于50%,密码子偏向使用A和U碱基并且以A和U碱基结尾。桃儿七5个叶绿体基因组IR/SC边界和基因组序列均比较保守。系统发育分析结果表明桃儿七和北美桃儿七亲缘关系最近。本研究获取了桃儿七5个叶绿体基因组信息与进化关系,为桃儿七资源开发利用与保护、品种鉴定和遗传进化研究奠定了科学基础。     

recO and recR mutations delay induction of the SOS response in Escherichia coli

RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage; therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of -galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.     

紫九牛叶绿体基因组密码子偏好性分析

为确定瑶药紫九牛叶绿体基因组密码子的使用模式及其成因,该研究以紫九牛叶绿体基因组50条蛋白质编码序列为研究对象,利用Codon W 1.4.2和在线软件CUSP和Chips分析其密码子偏好性。结果表明:(1)RSCU>1的密码子有29个,其中有28个以A/U结尾,说明叶绿体基因组的同义密码子中偏好以A/U结尾。(2)紫九牛叶绿体基因组密码子的GC含量GC₁(47.38%)>GC₂(39.81%)>GC₃(29.60%),ENC值大于45的有40个,说明紫九牛叶绿体基因组存在较弱的偏性。(3)中性绘图分析和ENC-plot分析说明了紫九牛叶绿体基因组密码子的偏好性既受到选择的作用,又受到突变因素的影响。(4)通过构建的高低基因表达库最终确定了15个最优密码子,分别为UUG、AUU、GUU、GUA、UCU、 CCU、ACU、ACA、GCU、CAA、AAC、GAA、UGU、CGU和GGU。该研究为紫九牛叶绿体基因组的确定以及遗传多样性分析提供了依据。     

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P₇₀₀ upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.     

蕨类植物叶绿体基因ycf94的分子进化研究

ycf94基因是近年来在叶绿体基因组中新发现的一个基因,在蕨类植物中表现高度保守。该研究共选取94种蕨类植物,在系统发育背景下,对ycf94基因的结构特征、密码子偏好性、进化速率和适应性进化进行分析。结果表明, ycf94基因的密码子偏好性较弱,偏好使用以A/U结尾的密码子,且不同物种间的偏好性存在一定差异。密码子偏好性的形成主要受到突变压的影响,同时也存在其他因素的作用;基于凤尾蕨科和其他蕨类中ycf94基因的结构特征存在区别,对两者的分子替换速率进行了比较,表明颠换率、非同义替换率和ω值间存在显著差异;仅检测出1个正选择位点74A,强烈的负选择作用表明ycf94基因的结构和功能基本趋于稳定。这为蕨类系统发育分析提供了新依据,并提供了解析ycf94基因功能的线索。     

A ribosomal frameshifting error during translation of the argl mRNA of Escherichla coli

Using fusions between the Escherichia coli genes argI and lacZ, it has been demonstrated that ribosomal frameshifting occurs at a frequency of between 3% and 16% within the argl mRNA, soon after the initiation codon. The frameshift involves a phenylalanyl-tRNA shifting into the + 1 frame at the sequence UUU-U/C. The shift does not occur if the in-frame phenylalanine codon UUU is replaced by UUC. The level of frameshifting is higher in dense cultures and is not dependent on phenylalanine starvation. In the wild-type argI gene this frameshifting event would be an error, leading to a truncated, non-functional protein. Therefore, it is unlike the numerous examples of required frameshifting events that have been described in other genes.     

拟南芥基因密码子偏爱性分析

密码子偏爱性对外源基因的表达强度有一定影响,特别是编码蛋白质N端7～8个氨基酸残基的密码子.通过对拟南芥染色体中26 827个蛋白质对应的基因密码子进行分析,得到了编码氨基酸的61种密码子在拟南芥中的使用频率,并与大肠杆菌和哺乳动物进行了比较,结果表明三者间的密码子偏爱性有较大差异.这一分析结果对于动物基因在植物中的表达,及植物基因在微生物中的表达具有一定指导意义.同时提供了一种直接以XML文档为数据源解析巨型XML格式染色体数据的方法.     

The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence and genetic expression in Escherichia coli

Summary Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agrecment with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The down-stream sequence exhibits homology with the 3 end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.     

A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli

A putative DNA glycosylase encoded by the Rv3297 gene (MtuNei2) has been identified in Mycobacterium tuberculosis. Our efforts to express this gene in Escherichia coli either by supplementing tRNAs for rare codons or optimizing the gene with preferred codons for E. coli resulted in little or no expression. On the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. Further comparison of the predicted mRNA secondary structures supported the hypothesis that mRNA secondary structure(s) surrounding the translation initiation region (TIR), rather than codon usage, played the dominant role in influencing translation efficiency, although manipulation of codon usage or tRNA supplementation did further enhance expression in the bicistronic vector. Addition of a cleavable N-terminal tag also facilitated gene expression in E. coli, possibly through a similar mechanism. However, since cleavage of N-terminal tags is determined by the amino acid at the P₁′ position downstream of the protease recognition sequence and results in the addition of an extra amino acid in front of the N-terminus of the protein, this strategy is not particularly amenable to Fpg/Nei family DNA glycosylases which carry the catalytic proline residue at the P₁′ position and require a free N-terminus. On the other hand, the bicistronic vector constructed here is potentially valuable particularly when expressing proteins from G/C rich organisms and when the proteins carry proline residues at the N-terminus in their native form. Thus the bicistronic expression system can be used to improve translation efficiency of mRNAs and achieve high-level expression of mycobacterial genes in E. coli.     

巨枝叶绿体基因组密码子偏好性分析

该文针对巨桉叶绿体基因组序列,选取其中长于300 nt且以AUG为起始密码子的43个非重复基因作为研究对象,采用CodonW1.4.2软件分析巨桉叶绿体基因组的密码子使用偏好性。结果表明:第3位密码子的平均GC含量为27.97%; ENC的变化范围为39.49～61.00,平均为47.04; RSCU1的密码子有31个,其中29个以A/U结尾;中性分析显示,GC12与GC3无显著相关;回归分析未达到显著性水平; ENCplot分析发现,大部分基因落在曲线上或附近;对应分析表明第1轴的贡献率为17.68%,第2轴的贡献率为11.49%,第3轴、第4轴的贡献率分别为8.00%和5.76%,前4轴累计贡献率达42.93%,第1轴与GC、ENC、CAI达到极显著相关。上述分析结果表明,巨桉叶绿体基因组的密码子偏好较弱,密码子第3位偏好以A或U结尾,选择和突变在巨桉叶绿体基因组密码子偏好中起相对均衡的作用,最终确定UUG、CUU、GUU、UCC、UCA、ACA、UAU、UAA、CAU、AAU、AGA和GGA 12个高频高表达密码子为最优密码子。这为转化叶绿体基因密码子优化,提高表达效率和改良巨桉目标性状奠定了坚实基础。     

毕赤酵母甲酸脱氢酶在大肠杆菌中的高表达及纯化

用PCR方法从毕赤酵母(Pichia pastoris)基因组DNA扩增甲酸脱氢酶(FDH)基因,通过定点突变密码子TAG(649-651位碱基)为GAG,突变后的基因片段插入表达载体pET-22b( )构建质粒pET-FDH,转化E．coli BL21(DE3)。基因工程菌在IPTG诱导下高效表达可溶性的FDH融合蛋白,蛋白的表达量占基因工程菌株总蛋白的30%。工程菌破壁上清液采用一步亲和层析分离,得到比活力为6．45U/mg的重组FDH。     

香樟转录组基因密码子偏好性分析

为了解香樟基因密码子偏好性,该文以NCBI网站中香樟转录组数据为材料,利用生物信息学手段评价转录组数据质量,选取高质量数据的转录组,去除低质量序列,组装转录组,预测基因结构,再利用自编perl脚本提取以AUG开头的基因序列37 Mb序列34 931个基因,进一步利用CodonW分析基因密码子偏好性。结果表明:GC含量的变化范围为0.273～0.742,均值为0.452; ENC的范围为26.29～61.00,均值为52.76; CAI的范围为0.064～0.401,均值为0.199; RSCU值大于1的密码子数目为27个,其中以U或A结尾的有22个; 中性分析表明,小部分基因在对角线上,大多数基因偏离对角线; ENC-plot分析表明小部分基因在标准曲线上,大多数基因偏离标准曲线。上述研究结果表明,香樟基因的密码子偏好性比较弱,密码子常以A/U结尾; 突变和选择两者都在密码子偏好中起作用,而选择作用更大; 最终确定了GUU、CAG、GAA、UCU、GCU、GGU为最优密码子,通过对目标基因密码子的校正,提高表达效率,从而为利用基因工程技术改良香樟重要性状奠定了基础。     

Lipooligosacc haridic antigens fromMycobacterium kansasii andMycobacterium gastri

A set of Lipooligosaccharides (LOSs) has previously been characterized inM. gastri W471. The structure of the highly antigenic LOS (LOS-III) was elucidated and this molecule can unambiguously distinguishM. gastri from the opportunistic pathogenM. kansasii. In the present study, the structures of three otherM. gastri W471 LOSs were determined by one-dimensional¹H-NMR spectroscopy and gas liquid chromatography. They differ by the number of Xylp units and by the structure of the distal monosaccharide. The two dimensional (2D) NMR approach was successfully applied to the LOS antigen ofM. kansasii to locate the acetyl and acyl substituents and to determine the anomeric configuration of the -d-Fucp unit.The molecular specificity of anti-LOS-III antibodies was investigated and the LOS-III epitope was defined as the distal disaccharide: 3,6-dideoxy-4-C-(1,3-dimethoxy-4,5,6,7-tetrahydroxy-heptyl)--xylohexp-(1 3)--l-Xylp.Abbreviations CI Chemical Ionisation - COSY ¹H-¹H COrrelated SpectroscopY - COSY LR ¹H-¹H COrrelated SpectroscopY Long Range - DQF-COSY Double Quantum Filtered¹H-¹H COrrelated SpectroscopY - EI Electronic Impact - GC Gas Chromatography - HMBC Heteronuclear Multiple Bond Correlation spectroscopy - HMQC Heteronuclear Multiple Quantum Correlation spectroscopy - HOHAHA HOmonuclear HArtmann-HAhn spectroscopy - HPLC High Performance Liquid Chromatography - ³J_H.H vicinal spin-spin coupling constants - LAM LipoArabinoMannan - LOS LipoOligoSaccharide - MS Mass Spectrometry - FAB/MS Fast Atom Bombardment-Mass Spectrometry - ¹H- or¹³C- NMR proton or carbon Nuclear Magnetic Resonance - PBS Phosphate Buffered Saline - PheG1 K-I Major phenolic glycolipids fromM. kansasii - RCT-1, -2, -3 relayed coherence transfer 1 step, 2 steps, 3 steps - ROESY Rotative frame Overhauser Effect SpectroscopY - Sugars Fucp: fucopyranose - Galp galactopyranose - Glcp glucopyranose - hexp hexopyranose - Rhap rhamnopyranose - Xylp xylopyranose - TLC Thin Layer Chromatography - TMS Tri Methyl Silyl     

Vererbungsstudien An Aphiochaeta xanthina Speis (Phoridae)

Zusammenfassung 1. Aphiochaeta xanthina Speiser (Phoridae) läßt sich leicht auf Drosophila-Nährboden züchten. Die Generationsdauer beträgt etwa 21 Tage. Die Art eignet sich bestens für Erbversuche.2. Eine Mutante mit heller Körperfarbe und Augenfarbe zeigt autosomalen Erbgang, eine andere mit heller Augenfarbe (occhi chiari) und eine mit reduzierter 4. Längsader dagegen partiell geschlechtsgebundenen Erbgang. In den Männchen findet zwischen dem Genort für occhi chiari und dem Geschlecht ein Austausch mit 1,13±0,25% Häufigkeit statt. 4. Längsader dagegen partiell geschlechtsgebundenen Erbgang. In den Männchen findet zwischen dem Genort für occhi chiari und dem Geschlecht ein Austausch mit 1,13 ± 0,25% Häufigkeit statt.     

Identification of a positive regulatory protein in Escherichia coli: the product of the cysB gene

Summary From the specialized transducing bacteriophage cysB, recombinant phages cysB242 and cysB257 have been obtained, each of which carries an amber mutation in the cysB cistron. A comparison of polyacrylamide gel electrophoretic profiles of labelled extracts from uv-irradiated bacteria that had been infected with cysB ⁺ or with cysB-amber phages, led to the identification of a 39,000-dalton polypeptide as the product of the cysB gene. The native protein was purified to near radiochemical purity and was found to be an oligomer with an isoelectric point close to pH 7.     

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after -ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5 and 3 ends of dTnp1 together with a perfect palindrome located after the 5 inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.