Sensitivity of Prestaining RNA with Ethidium Bromide Before Electrophoresis and Performance of Subsequent Northern Blots Using Heterologous DNA Probes

Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde–agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 μg/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to prestain RNA should not exceed 30 μg/mL. The efficiency of the hybridization of EtBr-prestained RNA was affected not only by factors that affect staining sensitivity but also by the type of probe used.     

Jeotgalibacillus aurantiacus sp. nov., a novel orange-pigmented species with a carotenoid biosynthetic gene cluster,isolated from wetland soil

A Gram-stain-positive, orange-pigmented, rod-shaped and flagellated bacterial strain T12^T was isolated from wetland soil in Kunyu Mountain Wetland in Yantai, China. The strain was able to grow at 15–40 °C (optimum 37 °C), at 0.0–9.0% NaCl (optimum 2%, w/v) and at pH 5.5–9.0 (optimum 8.5). A phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain T12^T is a member of the family Planococcaceae, sharing 97.6% and 97.1% sequence similarity with the type strains of Jeotgalibacillus salarius and Jeotgalibacillus marinus, respectively. Genome-based analyses revealed a genome size of 3,506,682 bp and a DNA G?+?C content of 43.7%. Besides, the genome sequence led to 55.0–74.6% average amino acid identity values and 67.8–74.7% average nucleotide identity values between strain T12^T and the current closest relatives. Digital DNA-DNA hybridization of strain T12^T with the type strains of Jeotgalibacillus proteolyticus and J. marinus demonstrated 19.0% and 20.3% relatedness, respectively. The chemotaxonomic analysis showed that the sole quinone was MK-7. The predominant cellular fatty acids were iso-C_15:0, anteiso-C_15:0, C_16:1ω7c alcohol and iso-C_14:0. The polar lipids consisted of an unidentified aminolipid, phosphatidylglycerol, diphosphatidylglycerol and two unidentified phospholipids. Based on the polyphasic characterization, strain T12^T is considered to represent a novel species, for which the name Jeotgalibacillus aurantiacus sp. nov. is proposed. The type strain is T12^T (=?KCTC 43296 ^T?=?MCCC 1K07171^T).

     

番茄潜叶蛾幼虫肠道可培养细菌结构组成及对大分子化合物的降解作用研究

番茄潜叶蛾Tuta absoluta是一种世界毁灭性番茄害虫。为明确其幼虫肠道可培养细菌的多样性及功能,本研究采用LB和NA两种培养基分别对番茄潜叶蛾幼虫肠道细菌组成进行了分离培养,根据细菌菌落形态和16S rDNA序列分析对细菌进行种属鉴定,采用比浊法测定了优势种的生长曲线,并采用透明圈法测定了肠道各可培养细菌对大分子化合物淀粉和纤维素的降解能力。结果表明,从番茄潜叶蛾3龄幼虫肠道中共分离到27株细菌,分属于3门10科17属24种,优势门、科、属、种分别是变形菌门Proteobacteria、欧文氏菌科Erwiniaceae、欧文氏菌属Erwinia、Erwinia iniecta,其相对多度分别达到90.68%、89.41%、89.41%和89.41%。优势种Erwinia iniecta在25℃,180 r/min的条件下培养无迟缓期,0~14 h为对数生长期,14~28 h为稳定期,28 h以后为衰亡期。Glutamicibacter属的L7和L9、考克氏菌属Kocuria的L14和短状杆菌属Brachybacterium的L20能同时降解淀粉和纤维素,考克氏菌属Kocuria的L15和L17只能降解淀粉,欧文氏菌属Erwinia的L、动性球菌属Planococcus的L11、微杆菌属Microbacterium的L18和Prolinoborus属的L22只能降解纤维素,其他菌株无淀粉和纤维素降解能力。综上所述,番茄潜叶蛾幼虫含有24种肠道可培养细菌,种类较为丰富,且部分细菌对淀粉和纤维素大分子化合物具有较强的降解作用,该结果将为番茄潜叶蛾肠道细菌多样性及其功能的深入研究提供依据,同时还为功能细菌的开发利用提供菌株。     

不同处理对濒危植物黄枝油杉扦插育苗的影响

黄枝油杉属珍稀濒危植物,树形高大,采种困难,母树开花结实少,种子发芽率低,繁殖速度慢。采用扦插育苗,方法简便易行,繁殖速度快。为此,针对与黄枝油杉扦插育苗成活、生长密切相关的插穗粗度、ABT生根粉浓度和施肥种类三大因素,分别进行对比试验。结果表明:插穗茎粗0.70cm用150mg/LABT生根粉溶液处理的平均成活率达95.6%;扦插成活后施用粪肥+尿素(配比为100∶0.4)的混合肥苗木生长健壮,平均抽梢长达30.8cm。方差分析和多重比较分析结果表明,插穗粗度、ABT生根粉浓度和肥料种类对黄枝油杉扦插苗木的生长影响极大。     

Las-like quorum-sensing system negatively regulates both pyoluteorin and phenazine-1-carboxylic acid production in Pseudomonas sp. M18

A las-like quorum-sensing system in Pseudomonas sp. M18 was identified, which consisted of lasI and lasR genes encoding LuxI-LuxR type regulator. Several functions of the las system from strain M18 were investigated in this study. The chromosomal inactivation of either lasI or lasR by recombination increased the production of both pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA) by 4－5 fold and 2－3 fold over that of the wild type strain of M18, respectively. Production of both antibiotics was restored to wild-type levels after in trans complementation with the wild-type lasI or lasR gene. Ex-pression of the translational fusions pltA׳-׳lacZ and phzA׳-׳lacZ further confirmed the negative effect of lasI or lasR on both biosynthetic operons, and it was also demonstrated that the las system was related to the ability of swarming motility and the inhibition of cell growth.     

Rad9 plays an important role in DNA mismatch repair through physical interaction with MLH1

Rad9 is conserved from yeast to humans and plays roles in DNA repair (homologous recombination repair, and base-pair excision repair) and cell cycle checkpoint controls. It has not previously been reported whether Rad9 is involved in DNA mismatch repair (MMR). In this study, we have demonstrated that both human and mouse Rad9 interacts physically with the MMR protein MLH1. Disruption of the interaction by a single-point mutation in Rad9 leads to significantly reduced MMR activity. This disruption does not affect S/M checkpoint control and the first round of G₂/M checkpoint control, nor does it alter cell sensitivity to UV light, gamma rays or hydroxyurea. Our data indicate that Rad9 is an important factor in MMR and carries out its MMR function specifically through interaction with MLH1.     

Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs. One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products. To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a P_ADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a P_HIS3-HIS3 cassette as a positive selection marker. This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid. To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin. A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B. The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.     

Hepatitis C virus 3′UTR regulates viral translation through direct interactions with the host translation machinery

The 3′ untranslated region (3′UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3′UTR and the host ribosome. The 3′UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3′UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3′UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation.