Characteristic features of thermal stability map of DNA in Escherichia coli and eukaryotic genes

Distribution of double-helix thermal stability of Escherichia coli and eukaryotic DNAs was analyzed. The results confirmed the previous propositions based on the study of the stability distribution in phage DNAs: (1) stability fluctuation appears near the boundaries of protein coding regions (PCRs) and non protein coding regions (NPCRs); (2) PCRs have less fluctuation than NPCRs. The present analysis also revealed that the local G + C content is lower in the beginning of PCRs of E. coli than the average G + C content of PCR and that deviations in the amino acid composition and the third letter usage PCRs are involved in the low G + C content; the biological meaning of this is discussed in relation to mRNA structure.     

Stability Distribution in the Phage λ-DNA Double Helix: A Correlation between Physical and Genetic Structure

Abstract

Statistical analyses on the positional correlation of physical-stability and base-sequence distribution maps with genetic map are made for the whole DNA (48502 bases) of λ-phage. The susceptibility to a double-helix unfolding perturbation and the fraction of the transient opening of a particular region of the double helix are adopted to define this physical stability.

The principal features obtained are: A) The DNA double strand of protein coding regions is found to have homostabilizing propensity around a defined stability which is characteristic to each individual gene. B) The stability of the double helix in non-protein coding region fluctuates, on average over the whole region, more than that in protein coding region. C) Boundary regions of protein coding and non-protein coding regions are regions of high stability-fluctuation. Stability especially fluctuates at the protein-coding-region side of the boundary. Contrary to the quiet feature of the interior part of protein coding region rather noisy part exists at its edge. D) One frequently opening region coincides with the attaching site for the site specific recombination between phage and bacterial DNA.

There are two possible ways to explain the noisy feature in the stability distribution in non-protein coding regions: 1) The region has been used as the locus of recombination as evolution took place. Thus DNAs which were homostabilized around a different value characteristic to each individual DNA, have been joined there many times, so that the noise has accumulated as a remnant of evolutional history; and/or 2) the base-composition homogenizing or double-helix homostabilizing mechanism does not work in unneeded region such as non-protein coding region or introns.

Since corresponding characteristics have been found in our previous analyses on other viral and globin-gene DNAs, the rules mentioned above may be comprehensively extended to other DNAs.     

Codon usage and base composition inRickettsia prowazekii

Codon usage and base composition in sequences from the A + T-rich genome ofRickettsia prowazekii, a member of the alpha Proteobacteria, have been investigated. Synonymous codon usage patterns are roughly similar among genes, even though the data set includes genes expected to be expressed at very different levels, indicating that translational selection has been ineffective in this species. However, multivariate statistical analysis differentiates genes according to their G + C contents at the first two codon positions. To study this variation, we have compared the amino acid composition patterns of 21R. prowazekii proteins with that of a homologous set of proteins fromEscherichia coli. The analysis shows that individual genes have been affected by biased mutation rates to very different extents: genes encoding proteins highly conserved among other species being the least affected. Overall, protein coding and intergenic spacer regions have G + C content values of 32.5% and 21.4%, respectively. Extrapolation from these values suggests thatR. prowazekii has around 800 genes and that 60–70% of the genome may be coding. Correspondence to: S.G.E. Andersson     

Molecular analysis of plasmid pAP4 from Acetobacter pasteurianus

The complete nucleotide sequence of plasmid pAP4 isolated from Acetobacter pasteurianus 2374^T has been determined. Plasmid pAP4 was analysed and found to be 3,870 bp in size with a G+C content of 50.1%. Computer assisted analysis of sequence data revealed 2 possible ORFs with typical promoter regions. ORF1 codes for a protein responsible for kanamycin resistance similar with Tn5 transposone, ORF2 encodes a resistance to ampicillin identical with Tn3 transposone. Plasmid has in A. pasteurianus five copies and in E. coli DH1 about 30 copies per chromosome and it segregation stability in both strains is very high. Based on the data on replication region, plasmid does not code for a replication protein and origin region is similar with ColE1-like plasmid.     

Two genes encoding gas vacuole proteins in Halobacterium halobium

Summary The archaebacterium Halobacterium halobium contains two related gas vacuole protein-encoding genes (vac). One of these genes encodes a protein of 76 amino acids and resides on the major plasmid. The second gene is located on the chromosome in a (G+C)-rich DNA fraction and encodes a slightly larger but highly homologous protein consisting of 79 amino acids. The plasmid encoded vac gene is transcribed constitutively throughout the growth cycle while the chromosomal vac gene is expressed during the stationary phase of growth. Comparison of the nucleotide sequences of the two genes indicates differences in the putative promoter regions as well as 35 single base-pair exchanges within the coding regions of the two genes. The majority of the nucleotide exchanges in the coding region occur in the third position of a codon triplet generating the codon synonym. The only differences between the two encoded proteins are the exchange of 2 amino acids (positions 8 and 29) and a deletion of 3 amino acids near the carboxy-terminus of the plasmid encoded vac protein. The genomic DNAs from other halobacterial isolates (Halobacterium sp. SB3, GN101 and YC819-9) were found to contain only a chromosomal vac gene copy. There is a high conservation of the chromosomal vac gene and the genomic region surrounding it among the halobacterial strains investigated.     

Gene content and density in banana (<Emphasis Type="Italic">Musa acuminata</Emphasis>) as revealed by genomic sequencing of BAC clones

The complete sequence of Musa acuminata bacterial artificial chromosome (BAC) clones is presented and, consequently, the first analysis of the banana genome organization. One clone (MuH9) is 82,723 bp long with an overall G+C content of 38.2%. Twelve putative protein-coding sequences were identified, representing a gene density of one per 6.9 kb, which is slightly less than that previously reported for Arabidopsis but similar to rice. One coding sequence was identified as a partial M. acuminata malate synthase, while the remaining sequences showed a similarity to predicted or hypothetical proteins identified in genome sequence data. A second BAC clone (MuG9) is 73,268 bp long with an overall G+C content of 38.5%. Only seven putative coding regions were discovered, representing a gene density of only one gene per 10.5 kb, which is strikingly lower than that of the first BAC. One coding sequence showed significant homology to the soybean ribonucleotide reductase (large subunit). A transition point between coding regions and repeated sequences was found at approximately 45 kb, separating the coding upstream BAC end from its downstream end that mainly contained transposon-like sequences and regions similar to known repetitive sequences of M. acuminata. This gene organization resembles Gramineae genome sequences, where genes are clustered in gene-rich regions separated by gene-poor DNA containing abundant transposons.Communicated by J.S. Heslop-Harrison     

Codon evolution is governed by linear formulas

When nucleotide (G, C, T and A) contents were plotted against each nucleotide, their relationships were clearly expressed by a linear formula, y = αx + β in the coding and non-coding regions. This linear relationship was obtained from the complete single-stranded DNA. Similarly, nucleotide contents at all three codon positions were expressed by linear regression lines based on the content of each nucleotide. In addition, 64 codon usages were also expressed by linear formulas against nucleotide content. Thus, the nucleotide content not only in coding sequence but also in non-coding sequence can be expressed by a linear formula, y = αx + β, in 145 organisms (112 bacteria, 15 archaea and 18 eukaryotes). Based on these results, the ratio of C/T, G/T, C/A or G/A one can essentially estimate all four nucleotide contents in the complete single-stranded DNA, and the determination of any ratio of two kinds of nucleotides can essentially estimate four nucleotide contents, nucleotide contents at the three different codon positions and codon distributions at 64 codons in the coding region. The maximum and minimum values of G content were ∼0.35 and ∼0.15, respectively, among various organisms examined. Codon evolution occurs according to linear formulas between these two values. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of Two C-P Bond-forming Enzymes Involved in the Biosynthesis of Bialaphos

An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158–163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 °C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 °C of HPH to 53 and 58.8 °C respectively. Specific activities and steady-state kinetics measured at 25 °C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions.     

First-neighbor frequencies of satellite DNAs from Drosophila nasutoides and Pagurus pollicaris from an analysis of their circular dichroism spectra

From an analysis of their circular dichroism spectra, we find that the four (A + T)-rich satellite DNAs of Drosophila nasutoides have distributions of first-neighbor base paris that resemble those previously found for other (A + T)-rich Drosophila satellites. We also apply our spectral analysis procedure for the first time to two (G + C)-rich satellite DNAs, those from the hermit crab Pagurus pollicaris. We find that P. pollicaris satellite I cannot be accurately analyzed with our standard set of spectral components and that P. pollicaris satellite II appears to be much like the synthetic polymer poly[d(A-G-C-)·d(G-C-T)] in its first-neighbor content.     

Nucleotide distribution in bacterial DNA's differing in G + C content

Summary The DNA's ofMicrococcus lysodeikticus andClostridium perfringens were fragmented to about 7 000 nucleotide pairs long by shear and fractionated with respect to buoyant density of mercury complexes in Cs₂SO₄. The distribution of G + C content in both DNA's was characteristically asymmetric. InM. lysodeikticus DNA, low G + C fragments were more numerous than high G + C fragments, whereas inC. perfringens DNA, high G + C fragments were more numerous than low G + C fragments. The G + C content of fragments ofM. lysodeikticus DNA varied from 70 to 77%, with a mean and standard deviation of 73.7 ± 1.92% G + C and that ofC. perfringens DNA varied from 27 to 34%, with a mean and standard deviation of 29.8 ± 1.34% G + C. The standard deviation was smaller than that ofEscherichia coli DNA fragments of similar size. Biological meanings of relatively low heterogeneity in nucleotide composition inM. lysodeikticus andC. perfringens are discussed.     

Nucleotide Sequence Determination and Genetic Analysis of theBacteroidesPlasmid, pBI143

The nucleotide sequence and genetic organization of theBacteroidesplasmid pBI143 were determined. The plasmid was 2747 base pairs (bp) and had a G+C content of 41% (GenBank Accession No. U30316). There were two open reading frames greater than 50 codons and these were designatedmobAandrepA.A 56-bp inverted repeat divided pBI143 into modules withrepAandmobAin separate regions. There was a marked difference in the G+C content and codon usage for the two regions;repAhad 33% G+C andmobAwas 44% G+C. MobA had homology to otherBacteroidesmobilization proteins and RepA shared homology to a replication protein fromZymomonas mobilisplasmid pZM2. These two putative replication proteins formed a subgroup of the rolling-circle replication proteins belonging to the pSN2 family of gram-positive plasmids. Consistent with this finding, single-stranded pBI143 DNA was detected in plasmid containingBacteroides fragiliscultures. Availability of the pBI143 sequence allowed the elucidation of the complete nucleotide sequence for pFD288 an 8.9-kbBacteroidesshuttle vector (GenBank Accession No. U30830).     

一株新型旱獭源性宽谱大肠杆菌噬菌体的分离鉴定及基因组分析

[目的]分离喜马拉雅旱獭肠内容物样本中的噬菌体,并研究其生物学特性和基因组特征。[方法]以大肠杆菌为宿主菌,利用双层琼脂平板法从喜马拉雅旱獭肠内容物样本中分离噬菌体;用透射电镜观察形态特征;测定其最佳感染复数、一步生长曲线、酸碱耐受度及宿主裂解谱等生物学特性,并进行全基因组测序。[结果]从喜马拉雅旱獭肠内容物样本中分离得到一株裂解性大肠杆菌噬菌体,命名为vB_EcoM_TH18,其噬菌斑呈无晕环的透亮圆形,透射电镜观察发现该噬菌体头部直径为(90±5) nm,尾部长度为(115±5) nm;最佳感染复数为1;一步生长曲线显示其潜伏期为10 min,110 min后进入平台期,平均裂解量为15 PFU/mL;在pH 4.5-9.5的范围内具有稳定活性;可裂解多种致病型和血清型大肠杆菌和宋内志贺氏菌,无法裂解沙门氏菌、屎肠球菌、金黄色葡萄球菌、肺炎克雷伯杆菌及鲍曼不动杆菌;基因组测序结果表明,其基因组长度为133 882 bp,GC含量为39.95%。基因组共注释到210个编码序列(CDS)和13个tRNAs,不含毒力基因及耐药基因。BLASTn比对结果表明该基因组与Avunavirus属噬菌体Av-05同源性为95.17%。基于噬菌体全基因组、主要衣壳蛋白和终止酶大亚基分别构建系统进化树,结果表明vB_EcoM_TH18是一株肌尾噬菌体科(Myoviridae) Avunavirus属的新型噬菌体。[结论]从喜马拉雅旱獭肠内容物中成功分离并鉴定了一株新型宽谱大肠杆菌噬菌体vB_EcoM_TH18,可裂解多种致病型和血清型的大肠杆菌及宋内志贺菌。     

Cooperative lengths of DNA during melting

The mean cooperative length of domains of DNA, determined from the variance in (G + C) content in derivative melting curves of large bacterial DNAs, varies from 230 base pairs (bp) for (A ? T)-rich domains to 580 bp for (G ? C) domains. These values correspond to values for the cooperativity parameter of 2(±2) × 10^?5 and 3(±2) × 10^?6, respectively, and to +7.2 and +9.6 kcal for the free energy of helix interruption in those regions.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

Phylogenetic Position of the Mitochondrion-Lacking Protozoan Trichomonas tenax,Based on Amino Acid Sequences of Elongation Factors 1α and 2

Major parts of amino-acid-coding regions of elongation factor (EF)-1α and EF-2 in Trichomonas tenax were amplified by PCR from total genomic DNA and the products were cloned into a plasmid vector, pGEM-T. The three clones from each of the products of the EF-1α and EF-2 were isolated and sequenced. The insert DNAs of the clones containing EF-1α coding regions were each 1,185 bp long with the same nucleotide sequence and contained 53.1% of G + C nucleotides. Those of the clones containing EF-2 coding regions had two different sequences; one was 2,283 bp long and the other was 2,286 bp long, and their G + C contents were 52.5 and 52.9%, respectively. The copy numbers of the EF-1α and EF-2 gene per chromosome were estimated as four and two, respectively. The deduced amino acid sequences obtained by the conceptual translation were 395 residues from EF-1α and 761 and 762 residues from the EF-2s. The sequences were aligned with the other eukaryotic and archaebacterial EF-1αs and EF-2s, respectively. The phylogenetic position of T. tenax was inferred by the maximum likelihood (ML) method using the EF-1α and EF-2 data sets. The EF-1α analysis suggested that three mitochondrion-lacking protozoa, Glugea plecoglossi, Giardia lamblia, and T. tenax, respectively, diverge in this order in the very early phase of eukaryotic evolution. The EF-2 analysis also supported the divergence of T. tenax to be immediately next to G. lamblia. Received: 15 February 1996 / Accepted: 28 June 1996     

改造大肠杆菌卟啉代谢途径对重组过氧化物酶活性的影响

【目的】原核表达某些需辅因子的外源蛋白时往往酶活偏低,为提高酶活和减少外加辅因子的成本,我们尝试在大肠杆菌中表达外源过氧化氢-过氧化物酶的同时提高大肠杆菌中与该酶辅因子相关的合成代谢。【方法】本研究克隆了中度嗜盐菌Halomonas elongata DSM2581的过氧化氢-过氧化物酶CAT-POD(catalase-peroxidase)编码基因kat G的ORF,构建原核表达载体p ET28a-kat G,实现了CAT-POD在大肠杆菌中的重组表达。由于CAT-POD活性依赖其活性中心血红素,而血卟啉是血红素的骨架,通过构建原核表达载体p UC19-tac-hem A,将编码5-氨基乙酰丙酸合成酶的hem A基因在大肠杆菌中过量表达,提高卟啉的含量,从而提高重组蛋白CAT-POD的酶活。【结果】最终的CAT酶活达到了377 U/m L,为对照组的7.5倍。【结论】本研究为工业生产高活性CAT-POD提供了有效的方案,也为体外重组表达含辅因子的蛋白提供可借鉴的思路。     

Stability distribution in the phage lambda-DNA double helix: a correlation between physical and genetic structure

Statistical analyses on the positional correlation of physical-stability and base-sequence distribution maps with genetic map are made for the whole DNA (48502 bases) of lambda-phage. The susceptibility to a double-helix unfolding perturbation and the fraction of the transient opening of a particular region of the double helix are adopted to define this physical stability. The principal features obtained are: A) The DNA double strand of protein coding regions is found to have homostabilizing propensity around a defined stability which is characteristic to each individual gene. B) The stability of the double helix in non-protein coding region fluctuates, on average over the whole region, more than that in protein coding region. C) Boundary regions of protein coding and non-protein coding regions are regions of high stability-fluctuation. Stability especially fluctuates at the protein-coding-region side of the boundary. Contrary to the quiet feature of the interior part of protein coding region rather noisy part exists at its edge. D) One frequently opening region coincides with the attaching site for the site specific recombination between phage and bacterial DNA. There are two possible ways to explain the noisy feature in the stability distribution in non-protein coding regions: 1) The region has been used as the locus of recombination as evolution took place. Thus DNAs which were homostabilized around a different value characteristic to each individual DNA, have been joined there many times, so that the noise has accumulated as a remnant of evolutional history; and/or 2) the base-composition homogenizing or double-helix homostabilizing mechanism does not work in unneeded region such as non-protein coding region or introns. Since corresponding characteristics have been found in our previous analyses on other viral and globin-gene DNAs, the rules mentioned above may be comprehensively extended to other DNAs.     

The relationship of DNA base composition and individual protein composition in micro-organisms

Summary To investigate the dependence of protein composition on DNA base composition, a set of data on individual proteins with known amino acid compositions from a spectrum of bacterial species has been compiled. It is found that similar relationships of amino acid frequency to G + C content exist for these proteins as for the bulk proteins studied by Sueoka (1961). The data are analysed by linear and cubic regression, and a measure of the proportions of A + T-rich and G + C-rich codons in the underlying messenger RNAs is put forward. The theoretical limits on the G + C content of coding DNA are discussed, and inference are made about the various selective forces acting on DNAs of different G + C contents.     

Synthesis of Two Polypeptide Subunits of an Aminoacyl tRNA Synthetase as a Single Polypeptide Chain

Abstract

E. coli aminoacyl tRNA synthetases are typically comprised of a single type of polypeptide chain. Glycine tRNA synthetase is an exception, and is comprised of two different subunits. Previous work showed that glyS encodes both subunits in a tandem arrangement of coding regions which are in the same reading frame. Nine nucleotides separate the TAA stop of the first coding segment (α-subunit) from the ATG start of the second one (β-subunit). A plasmid containing glyS was put into four different ochre suppressor strains. In three of them, significant quantities of an α-beta; fusion protein were synthesized in maxicells, in genetic backgrounds which retained cellular proteases. This shows that the fusion protein is stable in vivo and suggests that Gly-tRNA synthetase is operationally a single polypeptide which is the ancestor of the two subunits.