Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening

O⁶-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O⁶-methylguanine and O⁴-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined. When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein. With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.     

The problem with zeroes: why don't drosophilids lay eggs?

Summary We present some data on drosophilid oviposition and analyse the distribution of egg numbers over patches using an iterated negative binomial. This suggests that there are three different reasons for empty patches; patches are not found, patches are not suitable, or females are not able to lay eggs. This leads to five categories of site which can be disentangled using the iterated negative binomial. Since empty patches have important consequences for population dynamics and coexistence, this analysis will highlight how microscopic processes influence macroscopic behaviour in population biology.     

Nitrate reductases in hexaploid and tetraploid wheats and Aegilops

Summary Nitrate reductase activity (NR activity), protein content (NR protein) and polypeptides were compared in shoots of Triticum aestivum ssp. vulgare (L.) cv Fidel (bread wheat, AABBDD genome), Triticum dicoccum cv Vernal (AABB genome), Aegilops squarrosa var. strangulata (DD genome) and the amphiploid 365 (AABBDD genome), produced by crossing T. dicoccum cv Vernal and Ae. squarrosa var. strangulata. Constitutive NR protein and activity were found in shoots of all seedlings grown without nitrate, with the highest activity in the bread wheat. The inducible NR protein and activity developed upon the addition of nitrate. A 116-K polypeptide was identified as the main component of the NR from the bread wheat, while a faint, sometimes discernable 94-K band appeared on Western blots. Only one NR polypeptide could be identified in Ae. squarrosa —the 94 K. An intermediary situation was observed with the tetraploid T. dicoccum and the amphiploid: The 94-K polypeptide was the only one separated from NR of seedlings grown in the absence of nitrate. The 116-K polypeptide appeared after the addition of nitrate. The intensity of its band on the gel increased with the duration of the nitrate treatment. When comparing Ae. squarrosa and T. dicoccum, the constitutive isozyme (94-K polypeptide) was found in the D as well as in the AB genomes, while the inducible NR (116-K polypeptide) was absent from the D genome. Addition of the D genome into the AB genome slightly reinforced the expression of the inducible form (AB genome expression) in the amphiploid wheat. We postulate that the inducible form of NR in the bread wheat resulted from an evolutionary selection pressure favoured by cultivation.     

空气负离子对自发和环磷酰胺诱发的小鼠骨髓嗜多染性成红细胞微核率的影响

我们以前的研究表明负离子对~(60)Co-γ线诱发的急性和慢性损伤有明显的防护作用。本文以小鼠骨髓嗜多染性成红细胞(PCE)微核率为指标,探讨负离子对正常小鼠有无细胞毒性和对环磷酰胺(CP)诱发的遗传损伤有无防护作用。用负离子处理昆明小鼠60天后不注射CP直接取样;用负离子分别处理小鼠15天、30天和60天后注射CP,6小时后取样。镜检骨髓PCE微核并做统计处理。结果表明,负离子促进小鼠的生长发育且不导致潜在损伤;负离子对CP诱发的遗传损伤有明显的防护作用。     

The effects of drainage on groundwater quality and plant species distribution in stream valley meadows

Conditions in fen meadows in Dutch stream valleys are influenced by both deep (Ca²⁺-rich) and shallow (Ca²⁺-poor) groundwater flows. The distribution patterns of phreatophytic (groundwater-influenced) plant species showed distinct relationships with the distribution of different groundwater types. Large fluctuations in the ionic composition of groundwater were observed in the upper peat layers of drained areas. Ca²⁺-rich groundwater was replaced by precipitation water to a considerable depth (1–1.5 m). These fluctuations in groundwater composition were less pronounced in undrained areas.It was observed that characteristic species of fen meadow communities and rare phreatophytic species were restricted to areas with high water tables, which were nourished by either Ca²⁺-poor or by Ca²⁺-rich groundwater. Few species showed a preference for drained areas, where replacement of groundwater types gave rise to the occurrence of an intermediate groundwater type, which was thought to be the result of an increased acidification of the top soil (increased influence of infiltration water).It was argued that the endangered species were best preserved in areas with an undisturbed discharge of natural groundwater flows.Abbreviations EC = Electrical conductivity     

负二项分布与昆虫种群空间格局分析的研究现状

对农业有害生物及其天敌种群密度的正确估计是实施IPM(有害生物综合治理)方案的先决条件,因此,抽样方法一直被列为昆虫学,生态学和植物保护科学中最重要的基本     

Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells

Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay that is operational with endothelial cells, which mediate the cytoprotective effects of interest. We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of known and new PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation.     

Statistical test for detecting overdispersion in offspring number based on kinship information

In this paper, we develop a theory of a new statistic that tests overdispersion in offspring number on the basis of exactly known kinship relationships. The statistic utilizes the sample size and the number of kinship pairs found in a sample, specially the number of mother–offspring (MO) and maternal–half-sibling (MHS) pairs. Given a sufficiently large sample size, the statistic proposed in this paper approximately follows a standard-normal distribution under non-overdispersed conditions (Poisson’s variance). We found that (1) the value of the statistic (\(\ge 2\) or \(<2\)) reasonably indicates whether reproduction is overdispersed at the 5% significance level; (2) the power of the statistic is determined primarily by the balance between the degree of overdispersion and the sample size; (3) in many cases, if the number of kinship pairs can be approximated by a normal distribution, false-positive and false-negative situations can be avoided. The proposed method can detect moderate-weak levels of overdispersion that produce few MHS pairs in a sample because the effect of the population size (which determines the number of detected MHS pairs) is canceled by the detection of the number of MO pairs. Once the kinship determination procedure is established, this indirect measurement will be readily applicable to species even with weak overdispersion, expanding the available opportunities for understanding how overdispersion in offspring number affects ecological processes.     

杭州郊区菜蚜种群的空间动态

利用 1 990～ 1 992年杭州郊区菜区 1 0茬结球甘蓝、2 3茬小白菜上菜蚜种群的系统调查数据 ,选择受密度影响较小的负二项分布的K值 ,描述了菜蚜混生种群的空间格局及其时序动态 .2类蔬菜上桃蚜、萝卜蚜混生种群终年呈聚集分布 ,但聚集强度变化有明显的季节规律 .7～ 9月聚集度最高 ,5和 1 1月前后有 2个明显的扩散高峰 .在甘蓝作物上的聚集强度随时间的变化过程因季节而异 ,春夏季为高 低 高 ,夏秋季一直较高 ,秋冬季在年间有较大变化 ,冬春季开始由低向高变化 ,随后上下波动 .萝卜蚜的聚集度较桃蚜为高 .综合了菜蚜混生种群的数量消长规律、环境因子的变化等信息 ,对其空间图式及其时序动态特征的成因进行了讨论 .     

晋西北3个树种抗旱性指数的研究

根据对海红、刺槐和柠条的ＰＶ曲线主要水分参数的测定,进行了抗旱性指数季节变化和种间排序分析。结果表明,树种的抗旱性随着年生长发育节律和环境水分条件而改变,其抗早性生长季初最弱,夏季高温干早阶段增强,秋季降雨峰期减弱,生长季末最强或很强。柠条的抗旱性与海红很接近。这对黄土高原地区果树和饲料的开发与水土保持树种的推广有重要意义。