In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes

The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes.     

In vitro and in vivo leishmanicidal activity of Dysoxylum binectariferum and its fractions against Leishmania donovani

The leishmanicidal effect of crude ethanolic extract of stem bark of Dysoxylum binectariferum and its fractions has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Ethanolic extract was lethal to promastigotes as well as amastigote forms in macrophage system at the concentration of 100 microg/ml. Chloroform fraction significantly inhibited promastigote multiplication and was also active against amastigotes in infected J774A.1 macrophages at 100 microg/ml. Hexane fraction was moderately active and the other fractions were inactive against both the forms. When tested in vivo in hamsters, ethanolic extract was toxic at 500 mg/kg whereas exhibited marginal activity (67.7+/-5.3%) at 250 mg/kg x 5, p.o. on day 7 post treatment (p.t.) which increases slightly (69+/-4.7) by day 30 p.t. Chloroform and n-hexane fractions exhibited 64.3+/-4% and 47.8+/-4.6% parasite inhibition at the dose of 100 mg/kg x 5 p.o., respectively. The pure compound, rohitukine, obtained from chloroform fraction showed weaker in vitro activity and was ineffective in infected hamsters. The lead potential of this plant need further investigations.     

过表达Na,K-ATPase基因增强了黑曲霉对硅灰石的风化能力

【目的】探究和证实黑曲霉钠钾ATP酶(NKA)在硅灰石风化过程中的作用。【方法】以野生型黑曲霉(WT)为原始菌株构建黑曲霉Na,K-ATPaseα1基因(NKAα1)高表达菌株oeNKA。通过测定不同时间点(0d、2d、4d、6d)oeNKA和WT生物量、培养液pH值和矿物风化释放的Ca~(2+)浓度,并用X-ray diffraction (XRD)对风化后的矿物残渣进行检测,比较oeNKA和WT菌株对硅灰石这种硅酸盐矿物的风化效果。【结果】oeNKA菌株的NKAα1基因相对表达量和酶活分别为WT菌株的103倍和1.76倍。在持续6d的培养过程中,oeNKA与WT的菌丝体生物量变化趋势相同,在培养第2天时WT显著高于oeNKA,随时间差异逐渐缩小并在第6天达到最低;培养液pH值变化趋势相同,分别下降至3.64和3.87;oeNKA风化硅灰石时所释放Ca~(2+)浓度(1011.36±47.78μg/g)约为WT (248.30±25.21μg/g)的4倍;XRD检测图谱显示菌株oeNKA对硅灰石风化作用更明显。【结论】NKAα1过表达菌株oeNKA对硅灰石的风化能力显著高于WT菌株,且黑曲霉的NKA与硅灰石的风化有密切关联。     

微嗜酸寡养单胞菌中磷酸吡哆胺氧化酶基因pnpox生物学功能研究北大核心

【目的】研究微嗜酸寡养单胞菌(Stenotrophomonas acidaminiphila)CW117中磷酸吡哆胺氧化酶基因pnpox(phosphopyridoxamine oxidase,pnpox)在维生素B6(VB6)合成中的贡献及对黄曲霉毒素B1(aflatoxin B1,AFB1)的降解活性。【方法】采用基因插入突变方式,对菌株CW117中磷酸吡哆胺氧化酶基因pnpox进行突变,得到突变菌株。通过高效液相色谱法(high performance liquid chromatography,HPLC)检测突变株对AFB1的降解活性,以及突变株中吡哆醇和吡哆醛的合成情况,确定基因pnpox在寡养单胞菌体内VB6合成中的贡献和黄曲霉毒素降解代谢作用。【结果】成功构建了磷酸吡哆胺氧化酶基因突变子pnpox::pK19mobΩ2HMB,突变子吡哆醛的合成量较野生型菌株显著减少,吡哆醇合成量与野生型菌株无显著性差异;同时,突变子与野生型株CW117对AFB1的降解活性未发现显著性差异。【结论】菌株CW117中磷酸吡哆胺氧化酶在吡哆醛合成的过程中起着重要作用,该基因突变会导致VB6的严重缺乏,影响寡养单胞菌正常生长,但该基因对CW117降解黄曲霉毒素无显著性贡献。     

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.     

CRISPR-Cas9系统与mazF介导的大片段删减法在酿酒酵母染色体大片段删减中的比较

【目的】比较CRISPR-Cas9系统与maz F法这两种酿酒酵母染色体大片段删减方法。【方法】分别用上述两种方法删减了酿酒酵母长度为26.5 kb的染色体大片段YKL072W-YKL061W,并比较了两种方法的转化效率、敲除成功率。【结果】利用CRISPR-Cas9系统平均得到5个转化子,但正确率为100%;maz F法得到约100个转化子,正确率略低于前者,为93%。【结论】两种方法均能高效删减酿酒酵母染色体大片段,CRISPR-Cas9系统正确率较高,操作简便省时;maz F法相对稳定,对目的基因无PAM位点要求。     

罗布麻CesA基因家族的生物信息学分析

植物体中纤维素是细胞壁形成的主要成分,不仅参与细胞形态的建成,调控细胞发育,还参与细胞内多种细胞信号转导途径,进而影响植物体的生长发育。纤维素合酶是植物体合成纤维素的主要酶类。为了探究CesA基因家族对罗布麻生长发育及纤维素合成的调控机理,该文通过生物信息学分析方法,从基因家族鉴定、结构分析、蛋白理化性质与多级结构预测、亚细胞定位、信号肽、进化关系和顺式作用元件等方面,对罗布麻CesA基因家族进行系统鉴定和分子特征分析。结果表明:基于全基因组测序,罗布麻CesA基因家族鉴定含有15个成员,分布在罗布麻11条染色体中的8条上,其编码蛋白的氨基酸数量为730～1 158,相对分子质量81 280.81～130 123.18 kDa,理论等电点6.18～8.83。除了AvCesA3、AvCesA5、AvCesA7、AvCesA10和AvCesA11蛋白为稳定蛋白,其余成员均为不稳定蛋白;除了AvCesA12蛋白为疏水性蛋白,其余成员均为亲水性蛋白。该家族成员包含3～14个外显子,8～15个保守基序。编码蛋白主要分布于质膜与高尔基体上,无信号肽,二级结构以无规则卷曲与α-螺旋为主要构成元件。AvCesA15蛋白的跨膜结构域和三级结构与其他成员存在显著不同。罗布麻CesA基因进化时主要受纯化选择作用。对上游1 500bp区域顺式作用元件分析,结果显示罗布麻CesA基因受到光、温度、水分、氧气等环境因子及生长素、赤霉素、脱落酸、乙烯、水杨酸等植物激素调控。该研究为进一步探究罗布麻CesA基因家族的生物学功能、提高纤维品质与品种改良奠定理论基础。     

Molecular analysis of hprt mutations induced by chromium picolinate in CHO AA8 cells

Chromium picolinate (CrPic) is a popular dietary supplement, marketed to the public for weight loss, bodybuilding, and control of blood sugar. Recommendations for long-term use at high dosages have led to questions regarding its safety. Previous studies have reported that CrPic can cause chromosomal aberrations and mutations. The purpose of the current work was to compare the mutagenicity of CrPic as a suspension in acetone versus a solution in DMSO, and to characterize the hprt mutations induced by CrPic in CHO AA8 cells. Treatments of 2% acetone or 2% DMSO alone produced no significant increase in 6-thioguanine (6-TG)-resistant mutants after 48 h exposures. Mutants resistant to 6-TG were generated by exposing cells for 48 h to 80 μg/cm² CrPic in acetone or to 1.0 mM CrPic in DMSO. CrPic in acetone produced an average induced mutation frequency (MF) of 56 per 10⁶ surviving cells relative to acetone solvent. CrPic in acetone was 3.5-fold more mutagenic than CrPic in DMSO, which produced an MF of 16.2. Characterization of 61 total mutations in 48 mutants generated from exposure to CrPic in acetone showed that base substitutions comprised 33% of the mutations, with transversions being predominant; deletions made up 62% of the mutations, with one-exon deletions predominating; and 1–4 bp insertions made up 5% of the characterized mutations. CrPic induced a statistically greater number of deletions and a statistically smaller number of base substitutions than have been measured in spontaneously generated mutants. These data confirm previous studies showing that CrPic is mutagenic, and support the contention that further study is needed to verify the safety of CrPic for human consumption.     

Cre-LoxP系统在炭疽芽胞杆菌基因敲除中的应用及eag基因的敲除

将Cre-LoxP系统应用于Bacillus anthracis中并成功敲除eag基因．以B.anthracis基因组为模板扩增得到上下游同源臂,联合两端带有LoxP位点的壮观霉素抗性基因片段构建好同源重组载体,转化B．anthracis AP422,通过一系列筛选得到带有抗性标记的重组菌．然后,通过转入Cre重组酶表达质粒,去除抗性标记,得到eag基因缺失的重组菌,并在DNA水平、RNA水平和蛋白质水平进行了系统的鉴定．最终建立了Cre-LoxP系统在B．anthracis中的应用方法,并成功敲除eag基因.     

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991–1992 Liquidators than in Russian Controls (χ²=10.5, p=0.001) and in 1993–1994 Liquidators (χ²=8.3, p=0.004), and was marginally elevated relative to 1995–1996 Liquidators (χ²=3.3, p=0.07). This type of mutation has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991–1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p=0.009), not with increased MF in 1991–1992 Liquidators (p=0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p=0.0001), and also was elevated in Liquidators sampled in 1991–1992 (72%, p=0.0076), 1993–1994 (22%, p=0.037), and 1995–1996 (62%, p=0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.     

转录因子cpcR同源基因cpcR-c1和cpcR-t的功能鉴定

苏云金芽胞杆菌（Bacillus thuringiensis,Bt） LM1212菌株与典型的Bt菌株表型不同,可分化形成芽胞、形成细胞和晶体产生细胞。在LM1212菌株中,转录因子CpcR不仅参与了细胞分化过程,而且能够激活晶体蛋白基因cry35-like的启动子(P₃₅)。【目的】筛选cpcR同源基因,验证其生物学功能。【方法】本研究克隆了2个cpcR同源基因,来源于蜡样芽胞杆菌的cpcR-c1和来源于东洋芽胞杆菌的cpcR-t,将cpcR及其同源基因分别构建在pHT304-P₃₅-gfp、pHT304-P₃₅-lacZ报告载体上,获得的重组质粒转入无cpcR基因且无晶体蛋白基因的Bt HD73^–菌株中。利用激光共聚焦显微镜观察重组菌HD^–(cpcR-c1-P₃₅-gfp)和HD^–(cpcR-t-P₃₅-gfp)的细胞表型并进行芽胞计数实验。测定HD^–(cpcR-c1-P_35<...     

Mutation induction by different types of radiation at the Hprt locus

Mutation induction at the Hprt locus in Chinese hamster cells was studied after exposure to ultraviolet light, X-rays and alpha particles. While mutant frequency as a function of dose or fluence followed a linear–quadratic relationship with UV and X-rays, it showed a linear dependence for alpha particles. If mutant frequency is plotted vs. the logarithm of surviving fraction, a linear relationship is found in all cases although with different slopes. These are about equal with the two types of ionising radiations but about 10 times larger for UV. They can be used as a measure of mutagenic potential and are termed “mutagenicity”. It is shown that this parameter is correlated with the maximum of mutant yield, i.e., the number of mutants per cell at risk. It is concluded from this analysis that the maximum mutant yield is always found at doses or fluences which lead to 37% survival irrespective of the kind of radiation. If mutation induction is measured in X-irradiated cells after pre-exposure to UV, mutant frequency is higher than expected on the basis of independent action of the two radiations. Deletion spectra were determined by using multiplex polymerase chain reaction. It was found that the background of spontaneous mutants varied considerably and showed frequently repetitive patterns, presumably because of clonal expansion of pre-formed mutants. UV-induced mutants did not contain any deletions, while those with both X-rays and alpha particles the majority displayed partial and total deletions. Based on a total number of 134 X-ray- and 192 alpha-induced mutants, it is concluded that the total fraction of mutant clones without deletions (partial or total) is about 40% for X-rays and only about 20% for alpha-particles.     

地黄C3H基因的克隆及生物信息学分析

香豆酸-3-羟化酶属于植物中最大的蛋白酶细胞色素P450家族之一,在植物生命活动中发挥着重要作用。为了解地黄香豆酸-3-羟化酶基因RgC3H合成毛蕊花糖苷的功能,该研究基于地黄代谢组学分析获得KEGG途径中的C3H,采用多重比对在NCBI中获得同源基因的一个保守序列,并基于该保守序列和地黄SRA数据库,采用电子克隆和RT-PCR克隆技术获得地黄C3H基因全长CDS(RgC3H),对其进行生物信息学分析。结果表明:RgC3H基因全长为1 530 bp,且编码一个含509个氨基酸、分子量为57.91 kD、无信号肽的蛋白质; 基于氨基酸序列的结构分析显示,RgC3H有一个保守区域-P450结构域; 系统进化分析结果显示,RgC3H与芝麻和猴面花的C3H基因具有很高的同源性。上述结果为进一步研究RgC3H基因在地黄毛蕊花糖苷生物合成途径中的作用奠定了基础。     

柠条锦鸡儿F3H基因克隆及功能分析

柠条锦鸡儿为豆科灌木,对各种环境胁迫具有较强的适应能力,类黄酮是天然的抗氧化剂,花青素属类黄酮化合物,逆境胁迫会影响植物体内花青素的合成,而黄烷酮3-羟化酶(F3H)是花青素生物合成所必需的一种关键酶。该研究成功分离克隆了柠条锦鸡儿的F3H基因,命名为CkF3H。CkF3H基因的开放阅读框(ORF)为1095 bp,编码364个氨基酸,推测的蛋白质分子量为41.3 kDa,理论等电点为5.9。生物信息学分析发现,CkF3H基因序列与其它植物F3H有较高的一致性,推测CkF3H蛋白与其它植物F3H蛋白具有相似的功能。利用染色体步移法克隆得到CkF3H起始密码子ATG上游468 bp的启动子序列,PlantCARE软件分析表明,该序列具有启动子的基本元件CAAT-box和TATA-box以及多种与逆境胁迫相关的顺式调控元件。实时荧光定量PCR分析表明,CkF3H在柠条的根、茎和叶中均有表达,没有组织特异性;CkF3H的表达受低温、高盐、干旱和高温胁迫的诱导,并且在低温胁迫下,CkF3H的表达还受到光周期的影响。综上所述,研究结果表明CkF3H基因在柠条锦鸡儿适应低温、高盐、干旱和高温胁迫的过程中发挥作用。     

敲除aceE基因对猪链球菌丙酮酸代谢的影响

【目的】探究缺失编码丙酮酸脱氢酶蛋白的aceE基因对猪链球菌生长特性、三羧酸循环和丙酮酸代谢的影响。【方法】通过测量菌液的OD600值,绘制野生型菌株与aceE基因缺失突变株的生长曲线;利用试剂盒测定三羧酸循环和丙酮酸代谢旁路中乙酰CoA、琥珀酸CoA、延胡索酸、草酰乙酸、丙酮酸、乳酸和ATP的含量,通过荧光定量qRT-PCR确定柠檬酸合酶基因、苹果酸脱氢酶基因、琥珀酸脱氢酶基因、异柠檬酸脱氢酶基因、丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因的表达水平。【结果】与野生株相比,菌株ΔaceE在平台期OD600值下降;添加1g/L乙酸盐能够显著提升菌株ΔaceE平台期OD600值。菌株ΔaceE的丙酮酸含量上升,ATP含量下降;三羧酸循环代谢中乙酰CoA、琥珀酸CoA、延胡索酸含量降低;柠檬酸合酶基因和苹果酸脱氢酶基因表达水平上升,琥珀酸脱氢酶基因和异柠檬酸脱氢酶基因表达水平下调;在丙酮酸代谢旁路中丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因表达水平上升。【结论】结果显示,菌株ΔaceE三羧酸循环活性降低,虽然能够通过PDH旁路将部分丙酮酸分解为乙...     

巴西橡胶树HbSUT3和HbSUT5 mRNA在嫩茎和中脉中的原位杂交分析

为研究巴西橡胶树(Hevea brasiliensis)中HbSUT3和HbSUT5基因的功能,采用地高辛标记的RNA探针与橡胶树嫩茎和中脉两种组织切片分别进行RNA原位杂交,对这2种SUT基因在组织中的表达区域与表达特点进行了分析。结果表明,在橡胶树嫩茎中,两个SUT基因主要在树皮的韧皮部和皮层细胞中表达;在中脉中,两个SUT基因在除木质部导管系统外的其它部位均有表达;HbSUT3基因在嫩茎和中脉中的表达量相近,而HbSUT5基因在嫩茎中的表达量远高于中脉。这些揭示HbSUT3和HbSUT5基因可能广泛参与韧皮部装载、蔗糖运输与库细胞供给等活动,同时两个SUT基因也存在功能分化。     

The phylogenetic placement of the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.