In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA–protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5′-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5′-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2–36.1 kDa) DPCs is less efficient than that of an AP-peptide_10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.     

MICROTUBULES: EVIDENCE FOR 13 PROTOFILAMENTS

When microtubules are fixed in glutaraldehyde in the presence of tannic acid and thin sections cut, the subunit structure of the microtubule is readily observed without the need of image reinforcement. Seven types of microtubules were analyzed: those in the heliozoan axoneme, the mitotic apparatus, the contractile axostyle, repolymerized microtubules derived from the chick brain, the central pair in flagella, and the A tubules of flagella and the basal body. In all cases microtubules were composed of 13 equally spaced protofilaments. The B tubules in flagella and the basal body appear to be composed of 11 subunits. The connections of the B to the A and the C to the B are described. A model of a microtubule is presented.     

土壤食细菌线虫的原位富集培养方法

采用两种孔径的尼龙网袋（1mm和5μm）,将盆钵供试土壤分成内外两层,内层土壤混合猪粪或稻草,并以不添加猪粪和稻草的供试土壤作为空白;外层直接接入供试土壤,进行培养,以获取土著食细菌线虫大量富集的试验土壤。结果表明：添加基质（猪粪和稻草）显著地促进了土壤线虫的繁殖,大量繁殖的线虫通过1mm网袋迁移至外层未加基质的土壤,而采用5μm网袋则限制了线虫向外层土壤的迁移。添加猪粪的1mm网袋处理经过28d培养后,外层土壤线虫数是空白处理的9．1倍;添加稻草的1mm网袋处理经过35d培养后,外层土壤线虫数是空白处理的5．9倍。添加两种基质的5μm网袋处理,外层土壤线虫数和空白处理差异不大。添加基质主要是促进了食细菌线虫的繁殖,在培养结束时,添加猪粪的1mm网袋处理的食细菌线虫比例达到98．2％,添加稻草的1mm网袋处理的食细菌线虫比例达到90．5％,两个处理食细菌线虫的总数分别是空白处理的14．8倍和8．9倍,并且主要是Protorhabditis sp．线虫的增加。     

AFLP Phylogeny of 36 Erythroxylum Species

Four taxa of the plant genus Erythroxylum; Erythroxylum coca var. coca (Ecc), Erythroxylum coca var. ipadu (Eci), Erythroxylum novogranatense var. novogranatense (Enn) and Erythroxylum novogranatense var. truxillense (Ent) are cultivated primarily for the illicit extraction and processing of cocaine. Despite their economic and medical importance, the evolutionary history of these species remains unknown in a modern phylogenetic framework. The aims of this study were to: (a) investigate the relationship among the cultivated and a select number of non-cultivated taxa, and (b) test Plowman??s (Journal of Psychodelic Drugs 11:103?C117, 1979b) linear progression hypothesis of the cultivated Erythroxylum taxa versus Johnson??s et al. (Annals of Botany 95:601?C608, 2005) hypothesis that Ec and En are sister species. AFLP phylogeny was used to compare the relationships among 36 Erythroxylum species (133 accessions) spanning the geographic distribution of the genus. A Maximum Parsimony tree revealed both geographic and taxonomic partitioning into clades representing species from Africa, Asia-Pacific and the New World (Tropical Americas). Ec and En formed distinct clades, indicating they are sister species and a cluster of non-cultivated species were the most closely related to the cultivated species. Multivariate ordination analysis was used to evaluate the relationship between cultivated and non-cultivated Erythroxylum taxa from the Tropical Americas. Our results support the hypothesis that the cultivated species are more closely related to each other than to any other species of Erythroxylum, but refute the hypothesis that Ent (and Enn) descended from Ecc. Instead our data suggest an independent, non-linear evolutionary relationship between Ec and En. Finally, the AFLP analyses identified significantly different genetic groups within Erythroxylum suggesting that the current intrageneric classification of this genus be revised.     

Inability of Pseudomonas stutzeri denitrification mutants with the phenotype of Pseudomonas aeruginosa to grow in nitrous oxide.

Pseudomonas aeruginosa PAO1 reduced nitrous oxide to dinitrogen but did not grow anaerobically in nitrous oxide. Two transposon insertion Nos- mutants of Pseudomonas stutzeri exhibited the P. aeruginosa phenotype. Growth yield studies demonstrated that nitrous oxide produced in vivo was productively respired, but nitrous oxide supplied exogenously was not. The defect may be in electron transport or in nitrous oxide uptake.