Mutagenesis and carcinogenesis in nucleotide excision repair-deficient XPA knock out mice

Mice with a defect in the xeroderma pigmentosum group A (XPA) gene have a complete deficiency in nucleotide excision repair (NER). As such, these mice mimic the human XP phenotype in that they have a >1000-fold higher risk of developing UV-induced skin cancer. Besides being UV-sensitive, XPA^−/− mice also develop internal tumors when they are exposed to chemical carcinogens. To investigate the effect of a total NER deficiency on the induction of gene mutations and tumor development, we crossed XPA^−/− mice with transgenic lacZ/pUR288 mutation-indicator mice. The mice were treated with various agents and chemicals like UV-B, benzo[a]pyrene and 2-aceto-amino-fluorene. Gene mutation induction in several tumor target- and non-target tissues was determined in both the bacterial lacZ reporter gene and in the endogenous Hprt gene. Furthermore, alterations in the p53- and ras genes were determined in UV-induced skin tumors of XPA^−/− mice. In this work, we review these results and discuss the applicability and reliability of enhanced gene mutant frequencies as early indicators of tumorigenesis.     

Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids

Genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are generated both as byproducts of oxygen respiration or molecular executors in the host defense, and by environmental exposure to ionizing radiation and chemicals. To counteract such oxidative damage in nucleic acids, mammalian cells are equipped with three distinct enzymes. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-2'-deoxyguanosine triphosphate and 2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP), to the corresponding monophosphates. We observed increased susceptibility to spontaneous carcinogenesis in MTH1-null mice, which exhibit an increased occurrence of A:T-->C:G and G:C-->T:A transversion mutations. 8-Oxoguanine (8-oxoG) DNA glycosylase, encoded by the OGG1 gene, and adenine DNA glycosylase, encoded by the MUTYH gene, are responsible for the suppression of G:C to T:A transversions caused by the accumulation of 8-oxoG in the genome. Deficiency of these enzymes leads to increased tumorigenesis in the lung and intestinal tract in mice, respectively. MUTYH deficiency may also increase G:C to T:A transversions through the misincorporation of 2-OH-dATP, especially in the intestinal tract, since MUTYH can excise 2-hydroxyadenine opposite guanine in genomic DNA and the repair activity is selectively impaired by a mutation found in patients with autosomal recessive colorectal adenomatous polyposis.     

Convenient Procedure for Oligonucleotide-Directed in vitro Mutagenesis of Cloned DNA

Abstract

A new modification of the oligonucleotide-mediated mutagenesis technique has been used to produce specific base changes in the double-stranded plasmid DNA.     

PCR技术对人IL-3 cDNA进行定点突变的研究

本文利用PCR技术对人IL-3cDNA体外进行定点突变,将人IL-3cDNA第3位Met,第116位Lys密码子突变为Val密码子GTT。PCR扩增片段核苷酸序列与引物设计相应的cDNA突变体序列完全一致。结果证实此方法比经典寡聚核苷酸方法简单、迅速、成本低、效率高,也为基因的修饰,蛋白质工程研究提供了简便、稳定的方法。     

Analytical and structural electron microscopy of chromium carcinogenesis in vitro

Syrian hamster embryo cells have been grown in culture on thin (10–50 nm), evaporated substrates ofchromium, and examined byanalytical transmission electron microscopy. After 10 d exposure, significant metal uptake was observed and numerous cell colonies showed the physical characteristics ofcarcinogenic transformation. The majority of the ingested chromium appeared to be associated with nucleic acid complexes. As a control, cells grown on titanium showed no signs of metal uptake and had morphologies very similar to cells grown on conventional carbon substrates.     

基因体外诱变的一种新方法--脱氧核苷三磷酸替代法

在PCR的过程中,采用5-溴脱氧尿苷三磷酸(BrdUTP)部分取代脱氧胸苷三磷酸(dTTP)的方法,对克隆的野油菜黄单胞菌的α-淀粉酶基因进行了体外诱变.结果表明,BrdUTP浓度越高,诱变越强;浓度越低,诱变越弱.当BrdUTP浓度为dTTP的0.1%时,可以得到最多的正诱变结果.用LBSP鉴别培养基初筛,然后用Yoo改良法测定酶活,仅一轮诱变就获得了其表达产物α-淀粉酶的酶活分别降低了5倍和提高了20倍的两个突变基因.再以后者为PCR模板进行第二轮诱变,从而筛选到了α-淀粉酶的酶活提高40倍的突变体.此诱变方法克服了用碱基类似物在体内诱变由于核酸复制酶等的校正作用而造成诱变无效的难题,并为基因的体外诱变找到了一条新途径.     

绿玉树试管苗物理化学诱变及其抗寒突变体的筛选

以已建立的微体繁殖体系为基础,对绿玉树再生丛芽进行Co^60γ射线不同剂量的辐射和不同浓度EMS诱变处理,以HYP为突变体选择压,筛选出抗HYP突变体小苗,并对突变体小苗进行抗寒测试。结果表明：辐射剂量影响丛芽的增殖、突变率及芽苗的生长,1．5KR为绿玉树丛芽辐射诱变比较适宜的剂量。0．2％的EMS亦能诱导绿玉树丛芽块产生突变芽,但与正常培养获得的再生小苗相比,诱变后产生的抗HYP芽苗生长缓慢,再生小苗矮小。抗HYP突变小苗的抗寒性比正常植物的抗寒性强。     

应用PCR技术对先天性长QT综合征KCNQ1 基因进行定点突变的研究

利用聚合酶链反应（PCR）技术对长QT综合征（LQTS）KCNQ1基因进行定点突变的研究。首先设计两对引物（包含预定的突变）,通过3轮PCR扩增,扩增出含有所需突变位点的片段,然后将片段克隆入T载体中,通过酶切连接的方法将突变点引入到pIRES2-EGFP-KCNQ1中,随后用Effectene转染试剂介导转染HEK293细胞。结果在真核表达载体pIRES2-EGFP-KCNQ1基础上获得了KCNQ1 cDNA C934T的突变体,测序表明在序列中发生了预期的突变。将含突变点的pIRES2-EGFP-KCNQ1转染HEK293细胞后,在荧光显微镜下观察到被转染的HEK293细胞发出绿色荧光,表明含突变点的pIRES2-EGFP-KCNQ1得到了表达。Abstract: To study PCR site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro. The site-directed mutagenesis of LQTS gene KCNQ1 was made by PCR. Two sets of primers were designed according to the sequence of KCNQ1 cDNA, and mismatch was introduced into primers. Mutagenesis was performed in a three-step PCR. The amplified fragments from the third PCR which contained the mutation site were subcloned into the T-vecor PCR2.1.Then the fragments containing the mutation site was obtained from PCR2.1 with restriction enzyme digestion and was inserted into the same restriction site of pIRES2-EGFP-KCNQ1. With Effectene Transfection Reagent, pIRES2-EGFP-KCNQ1 was transfected into HEK293 cell. The sequencing analysis showed that the mutation site was correct. Mutation from T to C in 934 site of KCNQ1 cDNA was found. Under the fluorescence microscope, the green fluorescence was spread in the transfected HEK293 cell, meaning the pIRES2-EGFP-KCNQ1 containing the mutation site was expressed correctly.     

Mutagenesis of Neisseria meningitidis by in vitro transposition of Himar1 mariner

Now that the meningococcal genome sequence has been completed, the lack of a suitable method for saturation mutagenesis remains a major obstacle to the unraveling of the pathogenic propensity of Neisseria meningitidis. Here, we demonstrate that in vitro Himar1 mariner transposition on chromosomal or PCR-amplified meningococcal DNA, which is subsequently reintroduced into N. meningitidis by natural transformation, is an extremely efficient mutagenesis method. Southern blot analysis, sequencing the Himar1 insertion point in numerous transposition mutants, and a limited screening of the mutant libraries for clones impaired in maltose catabolism confirmed that Himar1 transposed randomly in N. meningitidis. Taken together, these data demonstrate that Himar1 in vitro transposition can lead to the exhaustive mutagenesis of N. meningitidis, allowing for the first time a genomic-scale mutational analysis of this important human pathogen.     

Barriers,nephrotoxicology and chronic testing in vitro

In many organs of the human body, there are effective physiological barriers which contribute to regulation of the uptake, transport and secretion of endogenous and exogenous materials. ECVAM is involved in the development of several in vitro models for detecting damage to various barriers, for example, the renal epithelium, the intestinal barrier, and the blood-brain barrier, after acute and chronic exposure to chemicals and products of various kinds. Long-term toxicity testing is an important issue in toxicology. At present, there are no generally accepted in vitro models available for replacing chronic testing in animals. Under chronic exposure conditions, the cellular response is larger than that which can be predicted in the standard cytotoxicity models. Therefore, the approach to predicting chronic toxicity will need to involve more-complex in vitro models. Several in vitro long-term toxicity systems currently available are under evaluation.     

兔肝细胞色素b5在大肠杆菌中的表达

利用体外特定点突变技术,将原核生物核糖体结合位点及十碱基间隔子(spacer)引科兔肝经胞色素b_5 cDNA的5′端,并构建了一个兔红血球b_5cDNA克隆,重组入表达载体pKK223-3质粒中,在大肠杆菌中成功地表达了兔肝和红血球两种形式的b_5蛋白。产量分别为每毫升培养液1.5μg和4.5μg。     

In vitro carcinogenesis studies on mouse fibroblast cells

Fibroblast cell lines were established from pulmonary explants derived from inbred CBA T6T6 mouse embryos. Cell lines controlled for the absence of spontaneous transformation were treated with 20=methylcholenthrene (0, 1 microgram/ml). The altered biological characteristics were studied during the process of the malignant transformation by the comparison of the untreated and 20-methylcholanthrene pretreated cell populations: the loss of contact inhibition and the connection between the malignant transformation and the arylhydrocarbon hydroxylase enzyme activity were investigated. No changes in the cell proliferation rate could be found following malignant transformation, but an increased resistance against altered circumstances was observed. In the course of passages, a gradual decreases in aryl hydrocarbon hydroxylase activity of the untreated line was seen, which disappeared or significantly decreased following 20-methylcholanthrene treatment, compared to the controls.     

Genotoxicity testing of fluconazole in vivo and in vitro

The genotoxic effects of the antifungal drug fluconazole (trade name triflucan) were assessed in the chromosome aberration (CA) test in mouse bone-marrow cells in vivo and in the chromosome aberration, sister chromatid exchange (SCE) and micronucleus (MN) tests in human lymphocytes. Fluconazole was used at concentrations of 12.5, 25.0 and 50.0 mg/kg for the in vivo assay and 12.5, 25.0 and 50.0 microg/ml were used for the in vitro assay. In both test systems, a negative and a positive control (MMC) were also included. Six types of structural aberration were observed: chromatid and chromosome breaks, sister chromatid union, chromatid exchange, fragments and dicentric chromosomes. Polyploidy was observed in both the in vivo and in vitro systems. In the in vivo test, fluconazole did not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increased in a dose-dependent manner compared with the negative control. The mitotic, replication and cytokinesis-block proliferation indices (CBPI) were not affected by treatments with fluconazole. According to these results, fluconazole is clastogenic and aneugenic in human lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of fluconazole.