Biased Usages of Arginines and Lysines in Proteins Are Correlated with Local-Scale Fluctuations of the G + C Content of DNA Sequences

Amino acid residues arginine (R) and lysine (K) have similar physicochemical characteristics and are often mutually substituted during evolution without affecting protein function. Statistical examinations on human proteins show that more R than K residues are used in the proximity of R residues, whereas more K than R are used near K residues. This biased use occurs on both a global and a local scale (shorter than ∼100 residues). Even within a given exon, G + C-rich and A + T-rich short DNA segments preferentially encode R and K, respectively. The biased use of R and K on a local scale is also seen in Saccharomyces cerevisiae and Caenorhabdidtis elegans, which lack global-scale mosaic structures with varying GC%, or isochores. Besides R and K, several amino acids are also used with a positive or negative correlation with the local GC% of third codon bases. The local-, or ``within-gene'-, scale heterogeneity of the DNA sequence may influence the sequence of the encoded protein segment. Received: 2 March 1998 / Accepted: 23 April 1998     

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.     

基于编码序列、基因间序列和氨基酸序列构建的系统发生关系比较

以7种古菌、46种细菌和10种真核生物的基因组为样本,考虑碱基间的短程关联和长程关联作用,得到编码序列的密码对和基因间序列的三联体对中不同位点的二核苷酸频率,据此构建了基于编码序列和基因间序列的系统发生关系。无论是基于编码序列还是基因间序列对信息进行聚类,古菌或真核均被聚在一支上,表明聚类参数的选择是合适的;与基于氨基酸序列构建的系统发生关系进行两两比较,发现大部分硬壁菌的编码序列与基因间序列之间,以及编码序列与氨基酸序列之间的进化都存在较大差异。通过分析认为,只有综合考虑这三类序列的进化信息,才可能得到更自然的系统发生关系。     

人类线粒体DNA变异的检测方法和思路

基于线粒体DNA（mtDNA)的研究对于人群源流迁移、线粒体相关疾病病因的探讨和法医鉴定等具有重意义,就检测人线粒体突变的一些常用方法,如RFLP、SSO和控制区测序等作一小结和归纳,并重点介绍目前mtDNA突变的筛选方法和思路,另外,还总结了近年来对人mtDNA方面的研究结果,对世界人群中主要单倍型类群（haplogroup)特征变异位点和相应的酶切检测引物作了归纳。     

Human Mitochondrial DNA Variation and Evolution: Analysis of Nucleotide Sequences from Seven Individuals

We have analyzed nucleotide sequence variation in an approximately 900-base pair region of the human mitochondrial DNA molecule encompassing the heavy strand origin of replication and the D-loop. Our analysis has focused on nucleotide sequences available from seven humans. Average nucleotide diversity among the sequences is 1.7%, several-fold higher than estimates from restriction endonuclease site variation in mtDNA from these individuals and previously reported for other humans. This disparity is consistent with the rapidly evolving nature of this noncoding region. However, several instances of convergent or parallel gain and loss of restriction sites due to multiple substitutions were observed. In addition, other results suggest that restriction site (as well as pairwise sequence) comparisons may underestimate the total number of substitutions that have occurred since the divergence of two mtDNA sequences from a common ancestral sequence, even at low levels of divergence. This emphasizes the importance of recognizing the large standard errors associated with estimates of sequence variability, particularly when constructing phylogenies among closely related sequences. Analysis of the observed number and direction of substitutions revealed several significant biases, most notably a strand dependence of substitution type and a 32-fold bias favoring transitions over transversions. The results also revealed a significantly nonrandom distribution of nucleotide substitutions and sequence length variation. Significantly more multiple substitutions were observed than expected for these closely related sequences under the assumption of uniform rates of substitution. The bias for transitions has resulted in predominantly convergent or parallel changes among the observed multiple substitutions. There is no convincing evidence that recombination has contributed to the mtDNA sequence diversity we have observed.     

Nucleotide Correspondence between Protein-Coding Sequences of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains 26695 and J99

Comparison of ORFs between H. pylori strains 26695 and J99 showed that transitions (more than 3%) prevail over transversions (less than 1%). The predominance of transitions was explained by the high rates of cytosine replacement by thymine in the coding (3.5–5.3%) and noncoding (2.9–3.9%) DNA strands. The proportion of transversion-type correspondences (A → C, A → T, C → A, C → G, G → C, G → T, T → A, and T → G) did not exceed 0.84%. The highest proportion (28.3%) was observed for correspondences between C and T in ACGT-ATGT, the target site of active methyltransferase of H. pylori J99 (M.Hpy99XI). It was assumed that C → T mutations due to cytosine methylation-deamination are prevalent in H. pylori.     

Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence is used as a template for repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recombination between diverged (“homeologous”) sequences. In this report, we have examined several aspects of HR between homeologous sequences in mouse and human cells. We found that gene conversion tracts are similar for mouse and human cells and are generally ≤100 bp, even in Msh2^−/− cells which fail to suppress homeologous recombination. Gene conversion tracts are mostly unidirectional, with no observed mutations. Additionally, no alterations were observed in the donor sequences. While both mouse and human cells suppress homeologous recombination, the suppression is substantially less in the transformed human cells, despite similarities in the gene conversion tracts. BLM-deficient mouse and human cells suppress homeologous recombination to a similar extent as wild-type cells, unlike Sgs1-deficient Saccharomyces cerevisiae.The ability of a cell to repair DNA damage is integral to maintaining genome integrity. One common type of damage that is particularly detrimental is a double-strand break (DSB), where both strands of DNA are broken. If not accurately repaired, DSBs can lead to cell death, chromosomal rearrangements, and loss of genetic material (reviewed in references 14 and 19). One mechanism of DSB repair is homologous recombination (HR), in which an unbroken homologous sequence, the donor of genetic information, is used as a template for repair of the broken sequence, the recipient of genetic information. HR intermediates possess heteroduplex DNA (hDNA), where one strand of DNA is derived from the donor sequence, and the second strand is derived from the recipient sequence. Mismatches in hDNA are substrates of the mismatch repair machinery (MMR) (reviewed in reference 38), leading to gene conversion. HR is the preferred repair pathway of DSBs in Saccharomyces cerevisiae (reviewed in references 42 and 46), plays an important role in repair of DSBs in Drosophila (1, 32), and is a major repair pathway of DSBs that occur during S/G₂ in mammalian cells (33, 54).Two pathways appear to predominate for the repair of DSBs by HR, both of which can give rise to noncrossover products, which predominate in mitotic mammalian cells (Fig. ​(Fig.1)1) (29, 52, 60). In the DSB repair model proposed by Szostak et al. (61), double Holliday junctions are resolved to result in recombinant products (Fig. ​(Fig.1A).1A). More recent evidence suggests the existence of an alternative pathway, termed synthesis-dependent strand annealing (SDSA) (Fig. ​(Fig.1B)1B) (20, 40, 42, 52). One difference between these two pathways is that the DSB repair model requires capture of both DNA ends (Fig. ​(Fig.1A),1A), which can lead to bidirectional gene conversion tracts. In contrast, SDSA can involve only one end of the broken DNA followed by dissociation (Fig. ​(Fig.1B),1B), resulting in predominantly unidirectional gene conversion tracts. Another difference is that the donor sequence can be altered during DSB repair while it typically remains unchanged after SDSA.Open in a separate windowFIG. 1.Models for noncrossover gene conversion resulting from DSB repair. DSB repair is initiated by resection of the DNA ends (black; strand directionality is designated a 3′ “tail”). The resected 3′ overhang invades the homologous donor template (gray), forming hDNA at the site of invasion (i), which acts as a primer/template for repair synthesis (gray dotted line). (A) In the canonical DSB repair (DSBR) model, the second strand of the DSB is captured, resulting in another stretch of hDNA (ii) and repair synthesis, to form a double Holliday junction. Depending on how the double Holliday junction is cleaved (arrowheads), resolution can result in a crossover (data not shown) or a noncrossover, as shown. (B) In SDSA, the newly synthesized strand dissociates from the D-loop and anneals to the other DNA end to form another stretch of hDNA (iii). Repair synthesis and ligation result in a noncrossover product. While one-end invasion is illustrated for the SDSA model, it is possible for both DNA ends to invade, resulting in gene conversion on both sides of the DSB (data not shown). In both models, hDNA formed by the newly synthesized strands can be repaired by MMR, resulting in gene conversion of markers (data not shown).HR repair is sensitive to differences between the recombining sequences, and cells have developed ways to suppress recombination between diverged sequences. This suppression of “homeologous” recombination reduces HR both between diverged repeats and with foreign DNA. Suppression of homeologous recombination is conserved across species and requires the MMR machinery (7, 10, 11, 49, 56). For example, MSH2 dramatically reduces both gene targeting (12) and DSB-induced HR (15) between sequences with >1% divergence in murine embryonic stem (ES) cells.Another protein that has been proposed to suppress homeologous recombination is Sgs1, the budding yeast RecQ helicase, as sequence divergence has little effect on recombination frequencies in Sgs1 mutants (39, 59). Sgs1 mutants have other phenotypes as well; for example, they demonstrate a hyperrecombination phenotype associated with spontaneous repair (22, 65, 68). The mammalian homolog of Sgs1 is BLM, mutants of which also have a hyperrecombination phenotype, as evidenced by a high frequency of sister-chromatid exchange (SCE) in both human and mouse cells (18, 24, 34, 69). Evidence suggests that Drosophila BLM, like Sgs1, has a role in the suppression of homeologous recombination (30) although mammalian BLM has not been tested in this regard. Supporting a possible role for BLM in suppressing homeologous recombination is the observation that BLM associates with MMR factors in a large protein complex (64; reviewed in reference 21), and BLM directly interacts with two components of the MMR machinery, MLH1 (45) and MSH6 (44), which, like MSH2, is known to suppress homeologous recombination (13).To gain more insight into mammalian HR mechanisms, as well as factors that control recombination between homeologous sequences, we examined recombination between homologous and homeologous sequences in both murine and human cells. By taking advantage of multiple, single base pair polymorphisms distributed along the donor in gene conversion substrates, we examined both the nature of gene conversion tracts and the fate of the donor sequence. Unidirectional tracts with a bias in conversion to one side of the DSB predominated in both mouse and human cells, supporting an SDSA mechanism of HR. Moreover, the donor remained unaltered after HR. Interestingly, while transformed human cells suppressed homeologous recombination, the degree of suppression was less than that observed in mouse cells. For either cell type, BLM deficiency did not alter this suppression, unlike what is observed in yeast Sgs1 mutants. Either other RecQ helicase family members play a role in the suppression of homeologous recombination, or mammalian RecQ helicases do not play a role in this process.     

Two Aspects of DNA Base Composition: G+C Content and Translation-Coupled Deviation from Intra-Strand Rule of A=T and G=C

The relative contribution of mutation and selection to the G+C content of DNA was analyzed in bacterial species having widely different G+C contents. The analysis used two methods that were developed previously. The first method was to plot the average G+C content of a set of nucleotides against the G+C content of the third codon position for each gene. This method was used to present the G+C distribution of the third codon position and to assess the relative neutrality of a set of nucleotides to that of the G+C content of the third codon position. The second method was to plot the intrastrand bias of the third codon position from Parity Rule 2 (PR2), where A=T and G=C. It was found that whereas intragenomic distributions of the DNA G+C content of these bacteria are narrow in the majority of species, in some species the G+C content of the minor class of genes distributes over wider ranges than the major class of genes. On the other hand, ubiquitous PR2 biases are amino acid specific and independent of the G+C content of DNA, so that when averaged over the amino acids, the biases are small and not correlated with the DNA G+C content. Therefore, translation coupled PR2-biases are unlikely to explain the wide range of G+C contents among different species. Considering all data available, it was concluded that the amino acid-specific PR2 bias has only a minor effect, if any, on the average G+C content. In addition, PR2 bias patterns of different species show phylogenetic relationships, and the pattern can be as a taxal fingerprint. Received: 5 November 1998 / Accepted: 1 March 1999     

不同方法建立的人大细胞肺癌裸鼠移植瘤模型的生物学特点

目的比较三种常用的皮下移植瘤造模方法建立的人大细胞肺癌NCI-H460裸鼠移植瘤模型的不同生物学特点,为不同的研究寻找合适的造模方法提供实验依据。方法分别用NCI-H460细胞,NCI-H460移植瘤组织块和移植瘤匀浆液对于BALB/c-nu/nu裸鼠建立皮下移植瘤模型,运用一般生物学指标观察三种移植瘤的成瘤率、瘤重、倍增时间和组织形态;采用全自动生化分析仪检测其外周血中丙氨酸氨基转氨酶（ALT）、天冬氨酸氨基转移酶（AST）、血糖（G1u）、尿素氮（BUN）和肌酐（CREA）等生化指标;利用血球分析仪检测白细胞总数（WBC）并进行分类,最后体外对其腹腔巨噬细胞吞噬活性和NK细胞活性进行了考察。结果本实验中细胞法和匀浆法的成瘤率及肿瘤生长速率显著高于埋块法且其生长更为均一,差异较小。接种5周后,与正常裸鼠比较,三组荷瘤小鼠血液中ALT、AST显著升高,BUN、CREA显著降低,埋块组的AST和BUN两项指标显著高于其他两荷瘤组。此外,接种2周后,荷瘤裸鼠的GLU显著低于正常裸鼠,匀浆液组的GLU降得最低。白细胞中,三种方法组荷瘤小鼠血液中LYM%、MN%、HGB均有降低,匀浆液组和细胞培养组的荷瘤小鼠血液中WBC、NEUT%、PLT显著高于埋块组。免疫细胞活性方面,两种细胞均呈现出正常细胞组〉匀浆组〉细胞组〉埋块组的趋势。结论细胞培养法接种数量可控,肿瘤生长均匀,适合建立不同实验需求的移植瘤模型,组织块移植法适于建立中药抗肿瘤筛选的动物模型,而匀浆液移植法则不推荐使用。裸鼠的生理生化状态和免疫功能与肿瘤的生长有密切的关系。     

Aligicidal nonfruiting myxobacteria with high G+C ratios

Summary The deoxyribonucleic acids of five algicidal nonfruiting myxobacteria are reported to have base ratios ranging from 69–71 G+C mole % as determined by thermal denaturation temperatures; no unusual nucleic acid bases were detected. These organisms are described as amicrocystogenous, gliding, Gram-negative bacilli capable of degrading gelatin, casein, starch, cellulose, chitin, and alginate. All have been shown previously to be algicidal. Poly--hydroxybutyrate in each was indicated by its conversion to crotonic acid. Antibiotic sensitivity of the five was similar to that of known nonfruiting myxobacteria. The fine structure of one, Myxobacter 44, revealed a triple-layered cellular envelope, the middle layer of which is lysozyme sensitive. Ruthenium red-positive slime material adhered to the outer surface.     

Characteristics of Neutral and Deleterious Protein-Coding Variation among Individuals and Populations

Whole-genome and exome data sets continue to be produced at a frenetic pace, resulting in massively large catalogs of human genomic variation. However, a clear picture of the characteristics and patterns of neutral and deleterious variation within and between populations has yet to emerge, given that recent large-scale sequencing studies have often emphasized different aspects of the data and sometimes appear to have conflicting conclusions. Here, we comprehensively studied characteristics of protein-coding variation in high-coverage exome sequence data from 6,515 European American (EA) and African American (AA) individuals. We developed an unbiased approach to identify putatively deleterious variants and investigated patterns of neutral and deleterious single-nucleotide variants and alleles between individuals and populations. We show that there are substantial differences in the composition of genotypes between EA and AA populations and that small but statistically significant differences exist in the average number of deleterious alleles carried by EA and AA individuals. Furthermore, we performed extensive simulations to delineate the temporal dynamics of deleterious alleles for a broad range of demographic models and use these data to inform the interpretation of empirical patterns of deleterious variation. Finally, we illustrate that the effects of demographic perturbations, such as bottlenecks and expansions, often manifest in opposing patterns of neutral and deleterious variation depending on whether the focus is on populations or individuals. Our results clarify seemingly disparate empirical characteristics of protein-coding variation and provide substantial insights into how natural selection and demographic history have patterned neutral and deleterious variation within and between populations.     

Few Single Nucleotide Variations in Exomes of Human Cord Blood Induced Pluripotent Stem Cells

The effect of the cellular reprogramming process per se on mutation load remains unclear. To address this issue, we performed whole exome sequencing analysis of induced pluripotent stem cells (iPSCs) reprogrammed from human cord blood (CB) CD34⁺ cells. Cells from a single donor and improved lentiviral vectors for high-efficiency (2–14%) reprogramming were used to examine the effects of three different combinations of reprogramming factors: OCT4 and SOX2 (OS), OS and ZSCAN4 (OSZ), OS and MYC and KLF4 (OSMK). Five clones from each group were subject to whole exome sequencing analysis. We identified 14, 11, and 9 single nucleotide variations (SNVs), in exomes, including untranslated regions (UTR), in the five clones of OSMK, OS, and OSZ iPSC lines. Only 8, 7, and 4 of these, respectively, were protein-coding mutations. An average of 1.3 coding mutations per CB iPSC line is remarkably lower than previous studies using fibroblasts and low-efficiency reprogramming approaches. These data demonstrate that point nucleotide mutations during cord blood reprogramming are negligible and that the inclusion of genome stabilizers like ZSCAN4 during reprogramming may further decrease reprogramming-associated mutations. Our findings provide evidence that CB is a superior source of cells for iPSC banking.     

Human DNA polymerase iota incorporates dCTP opposite template G via a G.C + Hoogsteen base pair

Human DNA polymerase iota (hPoliota), a member of the Y family of DNA polymerases, differs in remarkable ways from other DNA polymerases, incorporating correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. We present here the crystal structure of hPoliota bound to template G and incoming dCTP, which reveals a G.C + Hoogsteen base pair in a DNA polymerase active site. We show that the hPoliota active site has evolved to favor Hoogsteen base pairing, wherein the template sugar is fixed in a cavity that reduces the C1'-C1' distance across the nascent base pair from approximately 10.5 A in other DNA polymerases to 8.6 A in hPoliota. The rotation of G from anti to syn is then largely in response to this curtailed C1'-C1' distance. A G.C+ Hoogsteen base pair suggests a specific mechanism for hPoliota's ability to bypass N(2)-adducted guanines that obstruct replication.     

The Longest (A+T) and (G+C) Blocks in the Human and Other Genomes

Abstract

We analysed complete or almost complete nucleotide sequences of the human, chimp, mouse, rat, chicken, dog, and other genomes to find that they contain extremely long (A+T) a (G+C) blocks that do not occur at all in the corresponding randomized sequences. The longest is an (A+T) block containing 1040 consecutive AT pairs that occurs in the 16^th human chromosome. The longest human (G+C) block has 261 bp in length. About a half of the longest blocks occur in introns. The (A+T) blocks are discrete units whereas the (G+C) blocks are diffuse. They are embeeded in the genome through connectors longer than 1 kilobase where the (G+C) content gradually decreases to the value of 50%. Remarkably, the (A+T) as well as (G+C) blocks are substantially shorter in the chimp genome. Chicken is characteristic by very long (G+C) blocks that are even longer than in the human genome. Though much shorter, long (G+C) and especially (A+T) blocks occur in lower organisms as well, which means that AT and GC pair clustering is an ancient property that has evolved into large scales in higher eukaryote genomes and the human genome in particular. Very long (A+T) and (G+C) blocks confer specific biophysical properties on DNA that are likely to influence genome folding in cell nuclei and its functional properties.     

Mouse satellite DNA isolated by Ag+ -- CsSO4 density gradients contains G+C rich, slow reassociating sequences

Mouse satellite DNA sequences isolated by centrifugation in CS2SO4--Ag+ gradients are analyzed for buoyant density by CSCl density gradients and for their content of fast reassociating sequences by denaturation and partial reassociation. Our data suggest that in CS2SO4 gradients silver ions separate a satellite band which contains both fast reassociating G+C rich sequences and slow reassociating, A+T rich DNA sequences.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属（Eucalyptus）的10000条EST序列进行分析,在其中的1499条序列上共发现1775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外,还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关;微卫星的丰度与重复单元长度也呈负相关（三碱基重复微卫星除外）。在桉树EST序列中,重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面,桉树EST序列与杨树（Populus trichocarpa）基因组注释的转录序列随重复单元长度的变化呈现出相同的规律,但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低,推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计,并对设计的引物进行PCR检测,结果表明所设计的引物具有极高的扩增成功率。