Sequence of the lacI gene.

The structural gene for the lac repressor of Escherichia coli, the lacI gene has been sequenced. This 1,080 base pair region of the E. coli chromosome codes for the lac repressor protein of 360 amino acids. The DNA sequence largely confirms but extends the previously reported protein sequence and allows a structural analysis of genetic phenomena.     

Converting nonsense codons into sense codons by targeted pseudouridylation

All three translation termination codons, or nonsense codons, contain a uridine residue at the first position of the codon. Here, we demonstrate that pseudouridylation (conversion of uridine into pseudouridine (Ψ), ref. 4) of nonsense codons suppresses translation termination both in vitro and in vivo. In vivo targeting of nonsense codons is accomplished by the expression of an H/ACA RNA capable of directing the isomerization of uridine to Ψ within the nonsense codon. Thus, targeted pseudouridylation represents a novel approach for promoting nonsense suppression in vivo. Remarkably, we also show that pseudouridylated nonsense codons code for amino acids with similar properties. Specifically, ΨAA and ΨAG code for serine and threonine, whereas ΨGA codes for tyrosine and phenylalanine, thus suggesting a new mode of decoding. Our results also suggest that RNA modification, as a naturally occurring mechanism, may offer a new way to expand the genetic code.     

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.     

Protein evolution: causes of trends in amino-acid gain and loss

Understanding how proteins evolve is important for determining the molecular basis of adaptation, for inferring phylogenies and for engineering novel proteins. It has been suggested that some amino acids were incorporated into the genetic code more recently than others and, after comparing pairs of closely related genomes, Jordan et al. report that 'recent' amino acids are becoming more common. They argue that this process has been going on since the genetic code first evolved to encompass all 20 amino acids. Here we provide evidence that the patterns observed conform with standard, nearly neutral theoretical expectations and require no new explanation. This reinforces the need for caution in the interpretation of results derived from closely related taxa.     

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。     

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.     

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。     

连续小波变换在蛋白质结构预测中的应用

将代码为3pgal蛋白质的氨基酸序列映射为疏水值序列，在合适的尺度下，通过连续小波变换方法可对其非规则二级结构进行预测，淮确率为83．3％。从PDBsum数据库中随机抽取100个蛋白质作为测试对象，经本法处理后，1853个非规则二级结构中有1424个能被淮确预测到，平均预测淮确率为76．8％。结果表明：该法可较好地预测蛋白质的非规则二级结构，具有极大的发展前景。     

两种快速的密码子检索方法

许多生物信息学软件涉及将核苷酸序列翻译成对应的氨基酸序列的操作.密码子检索表的结构及检索算法强烈地影响蛋白质翻译的速度.本文提出了2种快速的密码子检索方法.一是平均检索长度约为7,最大检索长度为9的分块检索;二是没有空间冗余和地址冲突的哈希表检索,其平均检索长度和最大检索长度均为3.对几种密码子检索方法的检索效率进行比较,结果表明哈希表的检索速度最快.     

计算变异学创立背景及其用途

为了克服生物信息学和计算生物学中字母或数字不受序列长度、氨基酸组成和位置、相邻氨基酸影响的缺陷,根据自然界普遍存在的随机性原理,创立计算变异学。计算变异学用氨基酸对可预测性、氨基酸分布概率和变异概率3种方法量化整个蛋白质及每个氨基酸,用活的、动态的测量指标量化分析蛋白质。计算变异学方法可以应用于研究蛋白质进化、遗传病定量诊断,分析蛋白质结构与功能、药物设计和病毒变异预测等领域。     

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。     

一种新HDM2剪接变异体的cDNA克隆与序列分析

利用逆转录多聚酶链式反应(RT-PCR)分析癌基因HDM2在人宫颈癌Hela细胞中的表达.对克隆到的HDM2基因片段进行测序分析,从中筛选到一种新的HDM2剪接变异体.该剪接变异体阅读框由1 401 bp组成,预测编码466个氨基酸.与野生型HDM2相比,该变异体氨基酸序列的29-53位缺失,395住丝氨酸突变为苯丙氨酸,407位丝氨酸突变为半胱氨酸,其中缺失段29-53位于HDM2与p53相结合区域的上游部分.这可能影响HDM2与p53的相互作用,与p53的失活及癌细胞的转移相关.     

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.     

Aminoacylation of RNA minihelices with alanine

The genetic code is determined by both the specificity of the triplet anticodon of tRNAs for codons in mRNAs and the specificity with which tRNAs are charged with amino acids. The latter depends on interactions between tRNAs and their charging enzymes, and an advance in understanding such interactions was provided recently by the demonstration that a major determinant of the identity of alanine tRNA is located in the amino-acid acceptor helix. Multiple substitutions in many distinct parts of the molecule do not prevent aminoacylation with alanine. Substitution of the G3.U70 base pair with G3.C70 or A3.U70 in the acceptor helix prevents aminoacylation in vivo and in vitro, however, and the introduction of this base pair into tRNA(Cys) (ref. 1) or tRNA(Phe) (refs 1, 2) enables both to accept alanine. The importance of a single base pair in the acceptor helix and the results of recent footprinting experiments promoted us to investigate the possibility that a minihelix, composed only of the amino-acid acceptor-T psi C helix, could be a substrate for alanine tRNA synthetase. We show here that a synthetic hairpin minihelix can be enzymatically aminoacylated with alanine. Alanine incorporation requires a single G.U base pair, and occurs in helices that otherwise differ significantly in sequence. Aminoacylation can be achieved with only seven base pairs in the helix.     

小反刍兽疫病毒融合蛋白基因的克隆、序列分析及表达

根据GenBank报道的小反刍兽疫病毒(PPRV)融合蛋白(F)基因序列,用特异性引物对PPRV疫苗株F蛋白基因进行了RT-PCR扩增,并将其克隆到pGEM-T载体中进行测序。结果表明:F基因ORF全长1641 bp,编码546个氨基酸;推导的氨基酸序列中第1~18位氨基酸构成信号肽序列,第488~510氨基酸为跨膜区。构建原核表达载体pETF1和pETF2,转化E.coliBL21(DE3),用IPTG诱导表达。SDS-PAGE和Western-blotting的分析结果表明,F1和F2基因在大肠杆菌中均获得了表达,且均具有良好的反应原性。用Ni-NTA试剂盒纯化F1和F2重组蛋白,为研发检测PPRV特异性抗体的诊断试剂奠定了基础。     

一种考虑蛋白质柔性的分子对接方法

分子对接是计算机辅助药物分子优化设计中的一种重要方法,为此建立了基于诱导契合的分子对接优化模型.模型中引入残基基团的概念,将蛋白质划分成若干个残基基团,通过这些残基基团的运动近似表征整个蛋白质的运动情况,并将配体小分子的运动处理为平移、转动和柔性键旋转三部分分量.设计了一个将k-均值聚类和遗传算法相结合的快速迭代格式,并采用多种群遗传策略和信息熵控制的空间减缩搜索技术加速了分子对接设计中的遗传演化进程.在此基础上,开发了一种考虑蛋白质柔性的对接程序FkxGAsDock.数值试验表明,该程序较好地平衡了效率与精度之间的关系,取得了满意的对接结果.     

瘦素与能量平衡

通过对瘦素的研究分析,了解到瘦素受体广泛存在于下丘脑、肺、肾脏、肌肉和脂肪组织等中。瘦素是由ob基因编码、在脂肪组织合成分泌的蛋白质类激素,含有146个氨基酸,主要功能是调控进食、能量及体重。瘦素和其他激素一样,需要与特异的受体结合才能发挥其生物学作用。总之,瘦素的发现,为进一步研究肥胖的发生机制,以及预防和治疗肥胖开辟了一条崭新的途径。     

Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen.

DNA extracted from hepatitis B virus Dane particles has been cloned in bacteria using a plasmid vector. A full-length clone has been examined by restriction endonuclease analysis, and the nucleotide sequence of an 892-base pair fragment from cloned hepatitis B viral DNA encoding the surface antigen gene is reported. The amino acid sequence deduced from the DNA indicates that the surface antigens is a protein consisting of 226 amino acids and with a molecular weight of 25,398. The portion of the gene coding for this protein apparently contains no intervening sequences.     

tRNA丰度是影响蛋白质二级结构形成的一个因素

从人和大肠杆菌蛋白质和DNA序列的统计分析，发现mRNA的对应一定蛋白质二级结构的m密码子片段(m=3，4，…，8)，当平均tRNA拷贝数高时(对于人高于11，对于大肠杆菌高于2．O)，偏好编码螺旋，而避免编码线团；当平均tRNA拷贝数低时，偏好性正好反过来．对于beta折叠，未发现和tRNA拷贝数有明显的统计关联．对大肠杆菌也研究了密码子频数与蛋白质二级结构的相关性，其间的关联比tRNA拷贝数与蛋白质二级结构的关联弱．因此，tRNA拷贝数与蛋白质二级结构的关联可能更为本质．机制目前尚不清楚．一种可能的解释是：tRNA丰度以某种方式影响了新生肽链的折叠，通过翻译精度的因素影响了蛋白质二级结构的形成．