表皮生长因子受体基因突变检测方法改进及初步临床验证

目的:改进现有的检测表皮生长因子受体(EGFR)基因突变的荧光PCR法并开发出新的试剂盒,将其与直接测序法和ARMS法进行对比,验证该试剂盒用于临床诊断的敏感性、特异性和准确性。方法:收集2013年6月至2015年8月手术确诊的141例非小细胞肺癌(NSCLC)的石蜡包埋组织标本。采用盲法分别使用直接测序法、ARMS法和新试剂盒检测EGFR突变,比较新试剂盒与其他两种检测方法的差异,结果不一致时采用三种方法分别重复检验一次。结果:三种方法检测成功率均为100%,新试剂盒与直接测序法测得结果完全一致的比率达75.9%(107/141),在直接测序法测得的96例突变阳性中,92例在新试剂盒检测中得到验证(95.8%)。而直接测序法显示突变阴性的45例中,新试剂盒检测发现了23例突变阳性,两种检测方法的结果存在统计学差异(x2=40.745,P0.05)。与直接测序法进行比较,新试剂盒检测EGFR突变的敏感性、特异性分别为95.8%、48.9%,阳性预测值、阴性预测值分别为80.0%、84.6%,检测准确度为80.9%。以ARMS检测法为金标准,新试剂盒测得结果完全一致的比率达84.4%(119/141),两者的一致性比较好(K=0.749,P0.05),敏感性、特异性分别为94.1%、86.4%。结论:改进后EGFR基因突变检测的试剂盒在技术上较好地控制了检测结果的假阳性和假阴性,该检测方法较直接测序法具有更好的敏感性和准确性,与现有的ARMS法一致性较高。     

Strand targeting signal(s) for in vivo mutation avoidance by post-replication mismatch repair in Escherichia coli

Summary The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites. This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation. In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut ⁺) and deficient (mutHLS^-) strains. In mut ⁺ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold. Removal of the GATC site distal to the cat ^- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site. The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation. In the absence of a GATC site, mutL^- and mutS^- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH^- and mut ⁺ strains. This indicates that 50%–70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands. Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair     

Mechanisms of tetracycline and rifampicin resistance inBacteroides fragilis mutants obtained by ionizing radiation

Summary Based on a dose-survival curve, a radiation dose of 3.99 C/kg was used to induce antibiotic-resistant mutants inBacteroides fragilis. Escherichia coli B/r membrane fragments were employed as a reducing agent. Antibiotic-resistant mutants ofB. fragilis were utilized to study the mechanism by which these organisms become resistant to selected chemotherapeutic agents. Decreased accumulation of tetracycline by resistant mutants ofB. fragilis suggests that the resistance to this antibiotic is associated with the outer membrane permeability. There is a marked difference in the inhibitory action of rifampicin on RNA polymerase activity in rifampicin-sensitive and-resistant strains ofB. fragilis. This enzyme is, therefore, the likely target for inhibition of bacterial growth in this anaerobe by rifampicin.     

城镇污水诱发青蛙蝌蚪红细胞微核及其在环境监测中的应用

本文利用青蛙(Rana nigromaculata Hallowell)蝌蚪红细胞微核试验,作为检测城镇污水诱变活性的一种新的监测技术。在16d生活污水处理的实验中,青蛙蝌蚪红细胞微核细胞率2d后就呈现统计上的显著增加,并随处理时间的延长而增高,第12d达到最大值。在不同浓度混合污水处理实验中,蝌蚪红细胞微核细胞率呈现明显的剂量依赖性增加。上述实验证明城镇生活污水和混合污水都具有较强的诱变活性。作者从遗传毒理学的角度评价了湖北黄州综合生物塘系统对污水诱变活性的净化效能。城镇混合污水经综合生物塘各级塘处理,蝌蚪微核细胞率逐级下降,由进水的7.54‰下降到最后出水的1.52‰,接近对照(1.07‰)水平。其中综合生物塘的藻菌单元比水生植物单元对污水诱变活性具有更强的净化效力。本文提出污水“诱变指数”可作为综合生物塘一项功能评价指标。     

The cellular parameters of leaf development in tobacco: a clonal analysis

The cellular parameters of leaf development in tobacco (Nicotiana tabacum L.) have been characterized using clonal analysis, an approach that provides unequivocal evidence of cell lineage. Our results indicate that the tobacco leaf arises from a group of around 100 cells in the shoot apical meristem. Each of these cells contributes to a unique longitudinal section of the axis and transverse section of the lamina. This pattern of cell lincage indicates that primordial cells contribute more or less equally to the growth of the axis, in contrast to the more traditional view of leaf development in which the leaf is pictured as arising from a group of apical initials. Clones induced prior to the initiation of the lamina demonstrate that the subepidermal layer of the lamina arises from at least six files of cells. Submarginal cells usually divide with their spindles parallel to the margin, and therefore contribute relatively little to the transverse expansion of the lamina. During the expansion of the lamina the orientation and frequency of cell division are highly regulated, as is the duration of meristematic growth. Initially, cell division is polarized so as to produce lineages that are at an oblique angle to the midrib; later cell division is in alternating perpendicular planes. The distribution of clones generated by irradiation at various stages of development indicates that cell division ceases at the tip of the leaf when the leaf is about one tenth its final size, and then ceases in progressively more basal regions of the lamina. Variation in the mutation frequency within the lamina reflects variation in the frequency of mitosis. Prior to the mergence of the leaf the frequency of mutation is maximal near the tip of the leaf and extremely low at its base; after emergence, the frequency of mutation increases at the base of the leaf. In any given region of the lamina the frequency of mutation is highest in interveinal regions, and is relatively low near the margin. Thus, both the orientation and frequency of cell division at the leaf margin indicate that this region plays a minor role in the growth of the lamina.Abbreviation MF mutation frequency     

Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications

Summary We have obtained a revised estimate of the pattern of point mutation by considering more pseudogene sequences. Compared with our previous estimate, it agrees better with expectations based on the double-strand structure of DNA. The revised pattern, like the previous one, indicates that mutation occurs nonrandomly among the four nucleotides. In particular, the proportion of transitional mutations (59%) is almost twice as high as the value (33%) expected under random mutation. The same high proportion of transitions is observed in synonymous substitutions in genes. The proportion of transitional changes observed among electrophoretic variants of human hemoglobin is about the same as that predicted by the revised pattern of mutation. We also show that nonrandom mutation increases, by about 15%, the proportion of synonymous mutations due to single-nucleotide changes in the codon table, and increases, from 10% to 50%, the rate of synonymous mutation in the seven genes studied. However, nonrandom mutation reduces (by about 10%) the proportion of polar changes among nonsynonymous mutations in a gene. As far as single-nucleotide changes (in the codon table) are concerned, nonrandom mutation only slightly favors relatively conservative amino acid interchanges, and has virtually no effect on the proportions of radical changes and nonsense mutations.     

Free-radical-induced mutation vs redox regulation: Costs and benefits of genes in organelles

The prokaryotic endosymbionts that became plastids and mitochondria contained genes destined for one of three fates. Genes required for free-living existence were lost. Most genes useful to the symbiosis were transferred to the nucleus of the host. Some genes, a small minority, were retained within the organelle. Here we suggest that a selective advantage of movement of genes to the nucleus is decreased mutation: plastids and mitochondria have high volume-specific rates of redox reactions, producing oxygen free radicals that chemically modify DNA. These mutations lead to synthesis of modified electron carriers that in turn generate more mutagenic free radicals—the “vicious circle” theory of aging. Transfer of genes to the nucleus is also advantageous in facilitating sexual recombination and DNA repair. For genes encoding certain key components of photosynthesis and respiration, direct control of gene expression by redox state of electron carriers may be required to minimize free radical production, providing a selective advantage of organelle location which outweighs that of location in the nucleus. A previous proposal for transfer of genes to the nucleus is an economy of resources in having a single genome and a single apparatus for gene expression, but this argument fails if any organellar gene is retained. A previous proposal for the retention of genes within organelles is that certain proteins are organelle-encoded because they cannot be imported, but there is now evidence against this view. Decreased free radical mutagenesis and increased sexual recombination upon transfer to the nucleus together with redox control of gene expression in organelles may now account for the slightly different gene distributions among nuclei, plastids, and mitochondria found in major eukaryote taxa. This analysis suggests a novel reason for uniparental inheritance of organelles and the evolution of anisogametic sex, and may also account for the occurrence of nitrogen fixation in symbionts rather than in nitrogen-fixing organelles. Correspondence to: J.F. Allen