Molecular cloning and sequence analysis of the ribosomal ‘A’ protein gene from the archaebacterium,Halobacterium halobium

The ribosomal ‘A’ protein gene of Halobacterium halobium has been cloned and the nucleotide sequence of the DNA fragment containing the ‘A’ protein gene has been determined. The amino-acid sequence of the protein deduced from the nucleotide sequence was established from manual sequence analysis of the protein and structural data provided by peptides derived from cleavage of the protein with various proteinases. The ‘A’ protein consisted of 114 amino acids with a molecular weight of 11562 and was characterized mainly by a high amounts of alanine and acidic amino acid in the C-terminal half of the molecule. The coding sequence of the gene was preceded by a predicted Shine-Dalgarno sequence and two terminal codons. There was no intron or insertion sequence in the coding sequence. Following the terminal codon of the ‘A’ gene, there was a structure reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 71%. Inspection of the codon usage for the ‘A’ gene revealed 85% preference for G or C at the third codon position.     

甜瓜蔗糖转化酶基因的密码子偏好性分析

以甜瓜蔗糖转化酶基因序列为材料,研究甜瓜蔗糖转化酶基因密码子偏好性,为改良甜瓜风味与品质提供理论依据。运用在线分析软件Codon W对甜瓜蔗糖转化酶的编码序列(Coding sequence,CDS)进行密码子分析,利用Mobyle在线工具分析同义密码子相对使用度(RSCU)、有效密码子数(ENC)、GC及GC1s、GC2s、GC3s含量。甜瓜蔗糖转化酶基因偏好于以A或T结尾的密码子。密码子ATT、GTT和AGA的RSCU值都大于1,属于共同偏好使用的密码子,而密码子GCG、CGG的RSCU值小于1,属于使用频率较低的密码子。发现密码子偏好性与亲缘关系的远近有一定的关系。要实现目的基因在外源表达系统中的成功表达和提高其表达量,可通过增加目的基因剂量,目的基因密码子优化,改善培养条件等方法实现,其中目的基因密码子优化起到了关键作用。     

同义密码子用语的位置依赖

研究了在大肠杆菌编码区不同位置上的同底密码子用语,发现许多氨基酸的密码子用语在转译起始区有显著的变化,仅有少数氨基酸在转译区有较弱的变化,由于密码子用语与基因表达关系密切。这些结果与实验发现的编码区5‘端密码子用对表达的重要性是一致的。更进一步的结果还暗示了哪些密码子在特定位置的使用可能会影响基因表达。     

基于ND4和ND5基因序列分析的鳅超科鱼类系统发育关系

ND4和ND5是线粒体基因组中编码NADH脱氢酶亚基4和亚基5的两个蛋白质编码基因.该研究以鳅超科鱼类为研究对象,新测定了10个物种的ND4和ND5基因全序列以及中间的3个tRNA基因共212 bp的序列,结合从GenBank 下载的15个物种的15条序列进行序列比较和系统发育关系分析.结果显示:鳅超科鱼类ND4基因全长1380～1387 bp,以ATG为起始密码子,终止密码子为不完全终止信号;ND5基因全长1821～1839bp,同样起始密码子为ATG,终止密码子为TAA或TAG;ND4和ND5基因之间插入了3个tRNA基因,分别编码携带组氨酸、丝氨酸、亮氨酸的tRNA.ND4和ND5基因(包含3个tRNA基因)中A、T、G、C的平均含量分别为30.4%、27.3%、14.2%、28.1%,A+T(57.7%)的含量高于G+C(42.3%)的含量.转换与颠换比(Ti/Tv)平均值为1.586.选取斑马鱼和鲤鱼作为外类群,采用最大简约法(MP)、最大似然法(ML)和贝叶斯推断法(BI)进行系统发育树的重建.三种方法的系统发育分析结果都显示:花鳅亚科、条鳅亚科、沙鳅亚科、平鳍鳅科及Vaillantellidae分别构成单系;它们的系统发育关系为:(Vaillantellidae+(沙鳅亚科+(花鳅亚科+(条鳅亚科+平鳍鳅科).这与线粒体全基因组和某些核基因(如RAG1基因)的研究结果类似,且支持率较高,表明ND4和ND5基因用于鳅超科鱼类的系统发育分析是可行的;但是该研究的结果有别于其他线粒体基因的分析结果,如基于cytb和D-loop基因进行的系统发育分析表明,条鳅亚科和花鳅亚科聚为姐妹群,再和平鳍鳅科聚在一起.这种差异可能是由于使用的基因长度差异造成的,长度越长,信息量越大,所反映的系统发育结果可能更加接近真实情况.     

马铃薯密码子用法分析及其在t-PA基因密码子改造上的应用

采用bioperl-1.0 工具在红旗LINUX系统下自编了密码子分析软件;通过对马铃薯98个蛋白质编码基因序列（codon DNA sequence）的分析,计算出了马铃薯的密码子用法,并确定出了马铃薯的4个高表达优越密码子;依据马铃薯密码子用法和高表达优越密码子分析结果,对t-PA基因序列进行了密码子的改造,得到了具有马铃薯密码子使用特点的t-PA基因序列,从而为以马铃薯为生物反应器高效生产t-PA奠定了分子基础。 Abstract:Bioperl-1.0 was used under Hongqi LINUX system to programm the codon analysis software.According to the analysis of 98 codon DNA sequences with this software,the codon usage in potato was calculated and 4 codons have been inferred to the optimal codons.The codons of tissue plasminogen activator (t-PA) gene sequence have been reconstructed according to the results.The t-PA gene sequence containing the optimal codons of potato will be used for t-PA production by potato bioreactor.     

A frameshift addition causes silencing of the delta-globin gene in an old world monkey, an anubis (Papio doguera).

A delta-globin gene of an anubis, Papio doguera, was cloned, and the nucleotide sequence of a region of about 2,000 bp including the gene was determined. In comparison with that of pSP3, a delta-globin clone from the gene library of a normal Japanese, the anubis delta-globin gene showed one base addition in the second exon at the codon for amino acid 55, generating a termination codon at the new 59th codon. The other differences found in the region analyzed were not considered to affect expression of the delta-globin gene. Therefore, it was concluded that this frameshift addition was at least one of the causes of silencing of the delta-globin gene in the anubis. In addition, from the sequence divergence in the delta-globin gene, the period of this silencing was estimated to be at most 4 million years, and the divergence of anubis occurred at most 30 million years ago.     

Cooperative effects by the initiation codon and its flanking regions on translation initiation

The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.     

不同结构的蛋白编码基因的密码子偏性研究

利用聚类分析方法,对两类具有不同三级结构的75个蛋白的编码基因的密码子使用偏性进行了分析。75个基因样本序列按照对应蛋白的三级结构被很清晰的分成了两类,从而发现密码子的使用与蛋白质的三级结构有很大的相关性。这一重要结果证实了DNA的一维信息中蕴含着蛋白质的三级结构信息。     

Structure of the gene encoding the exoglucanase of Cellulomonas fimi

In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.     

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.     

Structural analysis of a beta-thalassemia gene found in Taiwan

The beta-globin gene from a patient with homozygous beta-thalassemia in Taiwan was cloned and extensively sequenced. Four nucleotides in the codon for amino acids, 41 and 42, are deleted. This change generates a frame-shift mutation and a termination codon in the new 59th codon. Some changes in nucleotide sequence were also found in intervening sequences IVS1 and IVS2, and Exon3, and were considered to be sequence polymorphisms.     

TTG as the initiation codon of Salmonella slyA, a gene required for survival within macrophages.

The slyA gene, which has been implicated in the virulence of Salmonella serovar Typhimurium and its survival in macrophages, is widely distributed among different Salmonella serovars. In this study, we cloned and sequenced the translational initiation region of the slyA gene from nine different serovars and found sequence differences in the previously proposed ATG initiation codon but not in a TTG triplet, another putative initiation codon in the slyA gene. Therefore, we determined the actual translational initiation site of the slyA gene by analyzing slyA genes with defined mutation in either the ATG or TTG sequences in an in vitro translation assay and a quantitative hemolytic assay in Escherichia coli. The replacement of TTG by TTC in the slyA gene significantly reduced both the amount of protein synthesized and the hemolytic activity of a transformed strain of E. coli, while replacement of ATG by ATC had no effect in these assays. In addition, the amino acid sequence analysis of the His-tagged SlyA protein showed that it was identical with the amino acid sequence deduced from the 5' end of the slyA gene with a TTG initiation codon. Our results suggest that TTG serves as the translational initiation codon for the slyA gene of Salmonella.     

The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase

The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.     

Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity

The complete nucleotide sequence of the Clostridium thermocellum celE gene, coding for an endo-beta-1,4-glucanase (endoglucanase E; EGE) with xylan-hydrolysing activity has been determined. The structural gene consists of an open reading frame (ORF) of 2442 bp commencing with a GTG start codon and followed by a TAA stop codon. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that derived by N-terminal amino acid sequencing of the purified protein. The EGE sequence contains a region homologous to the reiterated domain found at the C terminus of other endoglucanases from the same organism. BAL 31 deletions of the structural gene have revealed the extent to which this conserved sequence is necessary for endoglucanase and xylanase activity. A region of DNA, upstream from the structural gene has also been sequenced and a ribosome-binding site and putative promoter sequences have been identified. A second ORF which ends 349 bp 5' to the GTG start codon of the celE gene has also been identified. The encoded product contains a C terminus homologous to other C. thermocellum endoglucanases.