Characterization ofR. fredii QB1130, a strain effective on commercial soybean cultivars

Summary Two rhizobial strains (QB1130 and C3A) from northeast China were identified asRhizobium fredii on the basis of growth rate, media acidification and growth on a wide range of carbon substrates. The strains were shown to be distinct from USDA 191 on the basis of plasmid number and size. Bothnif and commonnod genes were located on the 295 kb plasmid of strains QB1130 and USDA 191, while onlynif genes were identified on this plasmid in C3A. When used to inoculate four commercial soybean (Glycine max) cultivars, one of the strains (C3A) was found to be ineffective, while the other (QB1130) was at least as effective as USDA 191, a strain ofR. fredii reported to be widely effective on North American cultivars of soybean. Further, QB1130 was capable of more effective nodulation of cowpea or the uncultivated soybean line, Peking, than either USDA 191 or the slow-growingBradyrhizobium japonicum USDA 16. Strain QB1130 should be useful for studies directed at improving symbiotic performance in soybean, or for studies of the comparative physiology and genetics of FG and SG strains on a single host.     

Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. IV. Comparison of dose-response curves for MNNG, 4NQO and ICR-170 induced inactivation and mutation-induction

The genetic effects of MNNG, 4NQO and ICR-170 have been compared on 5 different UV-sensitive strains and a standard wild-type strain of Neurospora crassa with regard to inactivation and the induction of forward-mutations at the ad-3A and ad-3B loci. Whereas all UV-sensitive strains (upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) are more sensitive to inactivation by MNNG and ICR-170 than wild-type, only uvs-5 shows survival comparable to wild-type after 4NQO treatment, all other strains are more sensitive to 4NQO. In contrast to the effects on inactivation, a wide variety of effects were found for the induction of ad-3A and ad-3B mutations: higher forward-mutation frequencies than were found in wild-type were obtained after treatment with MNNG or 4NQO for upr-1 and uvs-2, no significant increase over the spontaneous mutation frequency was found with uvs-3 after MNNG, 4NQO or ICR-170 treatment; mutation frequencies comparable to that found in wild-type were obtained with uvs-6 after MNNG, 4NQO or ICR-170 treatment and with upr-1 after ICR-170 treatment. Lower forward-mutation frequencies than were found in wild-type were obtained with uvs-2 after ICR-170 treatment and with uvs-5 after MNNG, 4NQO or ICR-170 treatment. These data clearly show that the process of forward-mutation at the ad-3A and ad-3B loci is under genetic control by mutations at other loci (e.g. upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) and that the effect is markedly mutagen-dependent.     

Integration of mammalian, microbial and drosophila procedures for evaluating chemical mutagens.

cis-Platinum(II)diamminodichloride (PDD), an anti-tumor agent, induced auxotrophic mutations in Escherichia coli, some of which were reverted to prototrophy by exposure to PDD, 2-aminopurine (2-AP), and N-methyl-N′-nitro-N-nitroguanidine (NTG), but not ICR derivatives. Similarly, various 2-AP-, NTG-, and ultraviolet light-induced auxotrophs were reverted to prototrophy by PDD. Some PDD-induced auxotrophs carried nonsense mutations and others could be phenotypically suppressed by growth with streptomycin. Although these findings suggest that PDD promotes base substitutions, this mutagen may also cause base subtractions because (like NTG)it induced, at reduced frequency, reversion to prototrophy of certain ICR-induced auxotrophs. Isomeric trans-platinum(II)diamminodichloride, which lacks anti-tumor activity, was an ineffective mutagen. Near-optimal conditions for PDD-induced mutagenesis entailed prolonged cultivation with low levels of mutagen where the frequency of forward mutation to auxotrophy was 10⁻³ and that of a selected trp isolate to prototrophy was 10⁻².     

miRNA-95靶向FOXD1基因对结直肠癌LoVo细胞放射敏感性的影响及其机制*

目的: 探讨miRNA-95靶向FOXD1基因对结直肠癌loVo细胞放射敏感性的影响及其机制。方法: 结直肠癌LoVo细胞购自武汉普诺赛生命科技有限公司,将LoVo细胞分为三组接种于6孔板中,每组3个复孔,实验重复3次,C组(对照组正常LoVo细胞),M组(miRNA-95-mimics转染LoVo细胞),I组(miRNA-95-inhibitions转染LoVo细胞),利用直线加速器6 MV/8 Gy的X射线垂直照射细胞,剂量率3 Gy/min,持续照射12 h后收集细胞。采用RT-PCR法检测LoVo细胞中FOXD1的表达情况,流式细胞术检测LoVo细胞凋亡情况,Western blot检测细胞中FOXD1蛋白的水平,细胞克隆测定细胞存活率,裸鼠成瘤实验取转染未经X射线照射的C组、M组、I组LoVo细胞细胞悬液,各取0.2 ml分别接种于C组、M组、I组BALB/C-nu雄性裸鼠左下肢足部皮上1次,每组10只,注射2周后,各组选取5只裸鼠进行X射线照射,每天4 Gy 照射一次,连续照射15 d,其余小鼠不照射,处死裸鼠并取出肿瘤进行称重。结果: 与C组比对,M组FOXD1水平明显升高,I组FOXD1水平明显低于M组、C组(P＜0.05);在同样8 Gy 照射后,与I组比对,C组和M组loVo细胞凋亡率显著下降(P＜0.05);在8 Gy照射后,与I组比对,M组和C组loVo细胞单克隆存活率有上升趋势(P＜0.05)。无照射条件下,与M组相比,C组和I组肿瘤体积减少(P均＜0.05),与C组比较,I组肿瘤体积减少(P＜0.05)。X射线照射15 d后,与无照射组各组裸鼠相比肿瘤体积显著减小(P均＜0.05),与M组相比,C组和I组肿瘤体积减少(P＜0.05)。结论: miRNA-95低表达可提高LoVo细胞的放射敏感性,促进细胞凋亡,其作用机制可能抑制FOXD1蛋白的活性有关。     

miRNA-191对前列腺癌的增殖、迁移侵袭能力的影响及其机制*

目的:观察miRNA-191对前列腺癌的增殖、迁移和侵袭能力的影响,并探讨其机制。方法:分别检测4种人前列癌细胞系(PC-3、DU-145、LNCa P、22RU1)及人正常前列腺细胞RWPE-2中miRNA-191的表达水平,并选择前列腺癌细胞系PC-3作为实验对象。将PC-3细胞分为3组:空白对照组(不转染)、miRNA-191 NC组(Inhibitor NC转染PC-3细胞)、miRNA-191 Inhibitor组(miRNA-191 Inhibitor转染PC-3细胞),每组设置3个复孔。采用RT-qPCR法检测PC-3细胞miRNA-191和PLCD1的mRNA表达水平;采用CCK8法检测PC-3细胞增殖水平;采用划痕实验和侵袭实验分别检测PC-3细胞迁移能力和侵袭能力;通过Targetscan靶基因预测网站,筛选PLCD1作为miRNA-191的靶向蛋白,并用双荧光素酶靶标实验验证;采用Western blot法检测PC-3细胞PLCD1的蛋白表达。结果:与RWPE-2细胞相比,人前列癌细胞中miNRA-191的表达水平显著升高(P＜0.05),且miRNA-191的表达水平在PC-3中较其他3种细胞系显著上调(P＜0.05)。抑制miRNA-191的表达水平后,PLCD1表达水平显著升高,PC-3细胞增殖能力受到抑制,迁移和侵袭能力较空白对照组和miRNA-191 NC组显著降低(P＜0.05)。双荧光素酶报告基因实验结果显示,PLCD1基因是miRNA-191的靶基因。结论:miRNA-191通过靶向PLCD1促进前列腺癌PC-3细胞的增殖、迁移和侵袭能力。     

人胰腺癌组织中miR-223与Hedgehog通路中SUFU、GLI1表达相关性分析

目的:探明SUFU、GLI1基因在正常胰腺组织以及胰腺癌和癌旁组织中的mRNA表达,分析miR-223的表达与GLI1、SUFU基因转录之间的关系,以及SUFU、GLI1基因在胰腺癌发病过程中的作用.方法:对正常胰腺组织以及胰腺癌和癌旁组织进行总RNA的提取,采用Real-time PCR方法,检测SUFU、GLI1基因的转录情况,对SUFU、GLI1的mRNA的表达量与miRNA-223的表达量进行相关性分析.结果:胰腺癌组织中GLI1 mRNA的表达量为4..79 (1.19,9.89),胰腺癌癌旁组织中的表达量为2.01(0.70,5.76)(P＜0.05).胰腺癌组织中SUFU mRNA表达量为1.34 (0.91,2.31),胰腺癌癌旁组织的表达量为1.51(1.23,2.56)(P＞0.05),胰腺癌癌旁组织中GLI1 mRNA的表达量与miR-223在胰腺癌癌旁组织中的表达量之间存在一定程度的相关性(P＜0.05).结论:GLI1mRNA参与了胰腺癌的发生,而SUFU mRNA与胰腺癌发生关系不大.GLI1基因及miR-223在胰腺癌的发生中可能起到一定的协同作用.     

Community structure,spatial distribution,diversity and functional characterization of culturable endophytic fungi associated with Glycyrrhiza glabra L.

A total of 266 endophytic fungal isolates were recovered from 1019 tissue segments of Glycyrrhiza glabra collected from four different locations in the North-Western Himalayas. The endophytes grouped into 21 genera and 38 different taxa. The host had strong affinity for the genus Phoma, followed by Fusarium. The species richness was highest at the sub-tropical location, followed by the sub-temperate location and the temperate locations, respectively. The tissue specificity of endophytes was also evident. Some endophytes showed potential antimicrobial activity against phyto-pathogens indicating that they may be helpful to the host in evading pathogens. All the endophytic taxa produced the plant growth promoting hormone, indole acetic acid (IAA), though in varying concentrations. None of these endophytes caused any symptoms of disease in co-cultivation with the tissue cultured plants. Further, all the endophytes had a positive influence on the phenolic and flavonoid content of the host. Three endophytes, Stagonosporopsis cucurbitacearum, Bionectria sp. and Aspergillus terreus also increased the host root (rhizome) and shoot growth visibly. Such endophytes are potential candidates for developing endophyte-based technologies for sustainable cultivation and enhanced productivity of G. glabra. This is the first report of community structure and biological properties of fungal endophytes associated with G. glabra.