Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see^1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.     

Involvement of separate pathways in the repair of mutational and lethal lesions induced by a monofunctional sulfur mustard.

The mutagenic and lethal effects of a monofunctional sulfur mustard, 2-chloro-ethylethylsulfide (CEES), have been studied in a number of repair deficient variants of Escherichia coli K12, B/r and B. The results indicate that CEES induces a (pre)mutational lesion which is subject to Uvr+-excision-repair. Extensive CEES-induced mutagenesis can occur in exrA- uvrA- and recA- uvrB- variants suggesting that the majority of the mutations in Uvr-bacteria do not arise from error-prone repair. These findings are similar to results previously reported with a volatile degradation product of captan and with ethyl methanesulfonate (EMS) but differ from those reported with methyl methanesulfonate (MMS). It is hypothesized that CEES alkylates guanine at the O-6 position (R-O-6-G) and that this R-O-6-G which is Uvr+-excisable is directly mutagenic by producing G-C to A-T transitions during replication. Reduced levels of induced mutation frequencies observed in an endonuclease II-deficient variant lead us to postulate that, in constrast to Uvr- bacteria, CEES-induced mutation in wild-type cells arise from error-prone repair of apurinic sites. Analysis of the lethal actions of CEES indicates that the lesion produced is largely unexcisable by the Uvr+ system. Host-cell reactivation of CEES-treated TI bacteriophage shows that the production of the (pre)ethal lesion is dependent on both the initial dose and post-treatment incubation. The efficient repair of the (pre)ethal lesion requires both endonuclease II and polymerase I. Moreover, deficiencies of these two enzymes rendered bacteria more sensitive to the cytotoxic action of CEES. It is postulated that the lethal mechanism of CEES involves: (I) alkylation at the N-3 position of adenine and the N-7 position of guanine; (2) spontaneous depurination of these alkylated bases; and (3) production of apurinic sites which are lethal unless repaired by the endonuclease II-polymerase I excision-repair system.     

Interaction of certain base-specific chemicals and diethylsulphate in the induction of chlorophyll mutations in Oryza sativa L.

The presoaked seeds of a rice cultivar, Tellakattera, were treated with three different concentrations of hydrazine (HZ) or hydroxylamine (HA) in combination with diethylsulphate (dES) (0.05%). In the M₁ generation more than additive effects were observed for increase in chlorophyll chimeras and decrease in seed fertility. A synergistic effect was also observed for both chlorophyll mutation and mutant frequencies, in the M₂ generation, in sequential treatments. However, the degree of synergism, based on M₂ chlorophyll mutant frequency, was greater in dES posttreatment combinations with HA or HZ, compared with dES pretreatments. These differences in reciprocal treatments may be due to more efficient fixation of premutational events by dES than HZ or HA.     

Genome assembly of the Chinese maize elite inbred line RP125 and its EMS mutant collection provide new resources for maize genetics research and crop improvement

Maize is an important crop worldwide, as well as a valuable model with vast genetic diversity. Accurate genome and annotation information for a wide range of inbred lines would provide valuable resources for crop improvement and pan-genome characterization. In this study, we generated a high-quality de novo genome assembly (contig N50 of 15.43 Mb) of the Chinese elite inbred line RP125 using Nanopore long-read sequencing and Hi-C scaffolding, which yield highly contiguous, chromosome-length scaffolds. Global comparison of the RP125 genome with those of B73, W22, and Mo17 revealed a large number of structural variations. To create new germplasm for maize research and crop improvement, we carried out an EMS mutagenesis screen on RP125. In total, we obtained 5818 independent M2 families, with 946 mutants showing heritable phenotypes. Taking advantage of the high-quality RP125 genome, we successfully cloned 10 mutants from the EMS library, including the novel kernel mutant qk1 (quekou: “missing a small part” in Chinese), which exhibited partial loss of endosperm and a starch accumulation defect. QK1 encodes a predicted metal tolerance protein, which is specifically required for Fe transport. Increased accumulation of Fe and reactive oxygen species as well as ferroptosis-like cell death were detected in qk1 endosperm. Our study provides the community with a high-quality genome sequence and a large collection of mutant germplasm.     

Effects of carcinogenic and non-carcinogenic chemicals on plasma esterases in BALB/c mice

Esterase profiles of plasma from female BALB/c mice treated with a variety of carcinogenic and weakly- or non-carcinogenic chemicals were analyzed. Mice treated with the potent carcinogens diethylnitrosamine, dinitrosopiperazine, dipropylnitrosamine, dimethylhydrazine, urethane, and dimethyldinitrosopiperazine had similarly altered plasma esterase profiles after 7 days' exposure to the chemicals. The alterations consisted of increased activity in 4 esterase bands. The increased activity persisted in some of the bands after cessation of carcinogen exposure. Exposure to high concentrations of the weakly- or non-carcinogenic compounds nitrosohydroxyproline, nitrosomethoxymethylamine, 1-nitroso-4 methylpiperazine, nitroso-2,6 dimethylpiperidine, and ethyl methanesulfonate caused no obvious plasma esterase alterations. Ingestion of carbon tetrachloride resulted in increased activity in one esterase band with concomitant decrease in a second band. Analysis of serum from test mice for levels of serum glutamic oxaloacetic transaminase, alkaline phosphatase, lactate dehydrogenase-lactate substrate, and D-gamma-glutamyl transpeptidase did not differentiate between mice treated with selected carcinogens and those treated with non-carcinogens and/or carbon tetrachloride.     

Functional complementation of the Arabidopsis thaliana psbo1 mutant phenotype with an N-terminally His6-tagged PsbO-1 protein in photosystem II

The Arabidopsis thaliana mutant psbo1 has recently been described and characterized. Loss of expression of the PsbO-1 protein leads to a variety of functional perturbations including elevated levels of the PsbO-2 protein and defects on both the oxidizing- and reducing-sides of Photosystem II. In this communication, two plant lines were produced using the psbo1 mutant as transgenic host, which contained an N-terminally histidine₆-tagged PsbO-1 protein. This protein was expressed and correctly targeted into the thylakoid lumen. Immunological analysis indicated that different levels of expression of the modified PsbO-1 protein were obtained in different transgenic plant lines and that the level of expression in each line was stable over several generations. Examination of the Photosystem II closure kinetics demonstrated that the defective double reduction of Q_B and the delayed exchange of Q_BH₂ with the plastoquinone pool which were observed during the characterization of the psbo1 mutant were effectively restored to wild-type levels by the His₆-tagged PsbO-1 protein. Flash fluorescence induction and decay were also examined. Our results indicated that high expression of the modified PsbO-1 was required to increase the ratio of PS II_α/PS II_β reaction centers to wild-type levels. Fluorescence decay kinetics in the absence of DCMU indicated that the expression of the His₆-tagged PsbO-1 protein restored efficient electron transfer to Q_B, while in the presence of DCMU, charge recombination between Q_A⁻ and the S₂ state of the oxygen-evolving complex occurred at near wild-type rates. Our results indicate that high expression of the His₆-tagged PsbO-1 protein efficiently complements nearly all of the photochemical defects observed in the psbo1 mutant. Additionally, this study establishes a platform on which the in vivo consequences of site-directed mutagenesis of the PsbO-1 protein can be examined.     

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.     

子宫内膜异位症的药物治疗

子宫内膜异位症(EMS)是妇科常见病,近年来发病率逐年上升,仅次于子宫肌瘤。虽然为良性疾病,却表现为恶性行为,主要特征为疼痛、不孕及性交困难,导致生活质量下降。其治疗目的在于消除病灶、缓解疼痛和解决生育问题,降低术后复发也不可忽视,因此,药物治疗尤为重要,不同的药物有不同的作用机理、副作用及用药时间。不同的患者需要根据其治疗目的、身体情况及经济状况选择不同的治疗方案。所以药物治疗应注意个体化、阶梯化和规范化。本文就近年来内异症的药物治疗做一综述。