Mutations induced by 60Co gamma-irradiation in double-stranded M13 bacteriophage DNA in nitrous-oxide saturated solutions are characterized by a high specificity

Irradiation of double-stranded M13 mp10 DNA in a diluted aqueous solution under N2O leads to a very specific mutation spectrum. Fifteen of 28 mutations induced in a 144 base pair (bp) target are C/G to G/C transversions, the other five bp substitutions are C/G to A/T transversions. Six mutations were single bp deletions, one is a large deletion of 180 bp and one is a 10 bp duplication which is probably from spontaneous origin. The mutations are not randomly distributed throughout the 144 bp mutation target but concentrated around two sites. The differences and similarities with the radiation-induced mutation spectrum previously obtained under oxygen are discussed.     

An application of PCR-single strand conformation polymorphism to MN genotyping

A PCR-based genotyping of MN blood group system was investigated for DNA samples taken from a population of 409 northern Japanese. DNA fragment (257bp) including exon 2 of glycophorin A (GPA) gene, in which encodes the determinants of MN antigens, was specifically amplified. On the analysis of PCR-single-strand conformation polymorphism (PCR-SSCP) for M alleles, band patterns of M(G) and M(T) were easily discriminated each other. For N alleles, three band patterns were observed, and we tentatively named these alleles as N(1), N(2) and N(V). The N(1) allele appeared predominantly and N(2) had two base substitutions at 1st (C-->A) and 56th (C-->T) in exon 2 of N(1). The other N(V), which was detected from a pair of a mother and her child, possessed a single base substitution at 23rd (A-->G) in intron 2. The allele frequencies of M(G), M(T), N(1) and N(2) were 0.4450, 0.0978, 0.4303 and 0.0269, respectively. The polymorphism information content and the probability of paternity exclusion by this MN genotyping were estimated to be 0.5252 and 0.3219, respectively.     

Dependence of the mutation spectrum in a shuttle plasmid replicated in human lymphoblasts on dose of gamma radiation.

The frequencies and types of mutations induced in the target gene, supF-tRNA, of the shuttle vector pZ189 were analysed following the replication of the gamma-irradiated plasmid in the human lymphoblastoid cell line, GM606. The mutation frequency measured in progeny of unirradiated pZ189 was 1.02 x 10(-4), increasing to 17.5 x 10(-4) at 1000 cGy, and to 63.4 x 10(-4) at 5000 cGy, approximately 17- and 62-fold over background levels, respectively. Simultaneously, the number of plasmids capable of replicating in Escherichia coli decreased with increasing radiation dose to 4% of the control value at 5000 cGy. Electrophoresis of the irradiated DNA showed a correlation between increases in mutation frequency and decreases in plasmid survival, and the formation of open-circular and linear DNA. The majority of the spontaneous (69.8%) and induced mutations (85.7%) at 1000 and 79.4% at 5000 cGy) were base substitutions and were generally of similar types among all groups. However, changes at 2500 (12.7%) and 5000 cGy (13.2%) involving A:T base pairs were greater than those in unirradiated controls (3.4%) or those at 1000 cGy (2.0%). This increase in A:T base pair mutations could be a result of reduced repair fidelity when the DNA is extensively damaged by high doses of ionizing radiation.     

山羊线粒体DNA细胞色素b基因序列分析

采用PCR直接测序法,对一个山羊群体线粒体DNA细胞色素b基因全序列进行测定。结果表明,山羊线粒体细胞色素b基因全长为1140bp,A、G、T和C碱基的平均含量分别为31.9%、13.1%、26.8%和28.2%,其中A T含量为58.7%,G C含量为41.3%,A T含量明显高于G C含量;密码子碱基的使用也存在一定差异。该研究为解决我国地方山羊品种起源系统中存在的分歧提供有益的探索。     

Mutagenic effects of 5-formyluracil on a plasmid vector during replication in Escherichia coli

Purpose : 5-Formyluracil (5-foU) is a major derivative of thymine produced in DNA by ionizing radiation and various chemical oxidants. It has been previously shown that 5-foU in template DNA directs misincorporation of nucleotides by DNA polymerases during in vitro DNA synthesis. The present experiments were designed to understand the biological effects of 5-foU in vivo. Materials and methods : The modified base was incorporated site-specifically into the recognition site of restriction endonuclease Sal I (5'-GTCGAC) or Afl II (5'-CTTAAG) in vector plasmid pSVK3 and introduced the plasmid into Escherichia coli. Results : When the plasmids were replicated in E. coli, 5-foU caused mutations at the target sites. The induced mutation frequencies were 0.038-0.049%. Sequence analysis revealed that 5-foU preferentially caused T:A →C:G and T:A →A:T base substitutions and -1 deletions at the 5-foU site. 5-FoU also caused mutations at sites near the 5-foU. The alkA mutation did not affect the frequency of mutations in 5-foU-containing plasmids. Conclusions : The present experiments demonstrated that 5-formyluracil in DNA caused mutations in E. coli.     

福建地区汉族人群骨保护素基因rs2073617T/C、rs2073618G/C多态性与急性冠脉综合征的相关性研究

目的 探讨骨保护素(OPG)基因启动子rs2073617T/C(950T/C)和第1外显子rs2073618G/C(1181G/C)位点基因多态性在福建地区汉族人群中的分布及其与急性冠脉综合征(ACS)的相关性.方法 纳入福建地区无血缘关系的汉族人720例作为研究对象,分为ACS组(n=360)和对照组(n=360),采用聚合酶链式反应－限制性片段长度多态性(PCR-RFLP)技术对OPG基因950T/C和1181G/C多态性位点进行基因型分型,同时采用DNA测序对酶切产物进行鉴定.结果 在福建地区汉族人群中,OPG基因950T/C多态性TC、TT、CC 3种基因型在ACS组和对照组中的频率分别为47.8％、26.7％、25.6％和43.3％、33.3％、23.3％;1181G/C多态性GG、GC、CC 3种基因型在ACS组和对照组中的频率分别为51.1％、40.0％、8.9％和60.0％、35.0％、5.0％.ACS组与对照组OPG基因950T/C、1181G/C基因型及等位基因频率分布进行比较均差异无统计学意义(P＞0.05).OPG基因950T/C、1181G/C在ACS组患者单支病变、双支病变及三支以上病变组之间比较差异无统计学意义(P＞0.05).结论 福建地区汉族人群OPG 950T/C、1181G/C位点基因多态性与ACS发生无相关性.     

Mutagenic effects of 5-formyluracil on a plasmid vector during replication in Escherichia coli

PURPOSE: 5-Formyluracil (5-foU) is a major derivative of thymine produced in DNA by ionizing radiation and various chemical oxidants. It has been previously shown that 5-foU in template DNA directs misincorporation of nucleotides by DNA polymerases during in vitro DNA synthesis. The present experiments were designed to understand the biological effects of5-foU in vivo. MATERIALS AND METHODS: The modified base was incorporated site-specifically into the recognition site of restriction endonuclease SalI (5'-GTCGAC) or AflII (5'-CTTAAG) in vector plasmid pSVK3 and introduced the plasmid into Escherichia coli. RESULTS: When the plasmids were replicated in E. coli, 5-foU caused mutations at the target sites. The induced mutation frequencies were 0.038-0.049%. Sequence analysis revealed that 5-foU preferentially caused T:A-->C:G and T:A-->A:T base substitutions and -1 deletions at the 5-foU site. 5-FoU also caused mutations at sites near the 5-foU. The alkA mutation did not affect the frequency of mutations in 5-foU-containing plasmids. CONCLUSIONS: The present experiments demonstrated that 5-formyluracil in DNA caused mutations in E. coli.     

Dependence of the Mutation Spectrum in a Shuttle Plasmid Replicated in Human Lymphoblasts on Dose of Gamma Radiation

Summary

The frequencies and types of mutations induced in the target gene, supF-tRNA, of the shuttle vector pZ189 were analysed following the replication of the gamma-irradiated plasmid in the human lymphoblastoid cell line, GM606. The mutation frequency measured in progeny of unirradiated pZ189 was 1·02 × 10^?4, increasing to 17·5 × 10^?4 at 1000 cGy, and to 63·4 × 10^?4 at 5000 cGy, approximately 17- and 62-fold over background levels, respectively. Simultaneously, the number of plasmids capable of replicating in Escherichia coli decreased with increasing radiation dose to 4% of the control value at 5000 cGy. Electrophoresis of the irradiated DNA showed a correlation between increases in mutation frequency and decreases in plasmid survival, and the formation of open-circular and linear DNA. The majority of the spontaneous (69·8%) and induced mutations (85·7% at 1000 and 79·4% at 5000 cGy) were base substitutions and were generally of similar types among all groups. However, changes at 2500 (12·7%) and 5000 cGy (13·2%) involving A:T base pairs were greater than those in unirradiated controls (3·4%) or those at 1000 cGy (2·0%). This increase in A:T base pair mutations could be a result of reduced repair fidelity when the DNA is extensively damaged by high doses of ionizing radiation.     

重性抑郁障碍患者与肿瘤坏死因子ɑ基因启动子区多态性的相关性研究

目的：探讨肿瘤坏死因子α（tumor necrosis factor,TNF－α）基因启动子区－308G／A、－857C／T位点多态性与重性抑郁障碍症发生的相关性。方法采用聚合酶链限制性片段长度多态性（ PCR－RFLP）分析方法检测393例重性抑郁障碍症患者和393名健康对照各个多态性位点的基因型,采用SPSS 13.0进行统计学分析。结果 TNF－α基因启动子－857 C／T位点的等位基因和基因型频率分布在正常对照组与重性抑郁障碍症组间存在统计学意义（ P＜0.05）。其中,男性重性抑郁障碍症组与对照组在－857 T的等位基因频率上存在显著差异（ OR＝0.31,95％CI：0.22－0.44,P＜0.01）,而女性病例组与对照组在－308 A位点的等位基因频率存在显著性差异（ OR＝0.40,95％CI：0.25－0.65,P＜0.01）。结论研究证实TNF－α基因启动子区多态性可能是中国北方汉族重性抑郁障碍患者的风险因素。 TNF－α基因启动子区多态性可能与重性抑郁障碍的发病存在关联。     

噬菌体表面展示技术筛选乙型肝炎病毒前S1蛋白结合蛋白基因

目的 以乙型肝炎病毒(HBV)前s1蛋白为靶蛋白,寻找其相应的结合蛋白。方法 应用T7 cDNA文库噬菌体展示技术克隆与前s1蛋白具有结合作用的蛋白序列,命名为前s1结合蛋白(PreS1BP)。将推断的氨基酸序列在蛋白质库中进行搜索,自GenBank中获得结合蛋白的cDNA和基因组的全长序列。结果 初步确定PreS1BP即为神经胶质瘤抑制基因区候选基因2(GLTSCR2),cDNA长为1436核苷酸,基因位于第19号染色体长臂13.3区(19q13.3),长度11445碱基对(bp),位于19号染色体10403483～10414989,PreS1BP基因含有13个外显子,具有12个内含子。结论 应用T7 cDNA文库噬菌体展示技术和生物信息学方法获得了HBV PreS1BP基因。     

军事噪声性听力损失的线粒体基因变异分析

目的研究线粒体基因变异与军事噪声性听力损失遗传易感性的关系,为确定噪声易感个体的分子诊断方法提供依据。方法对云南某部接触军事噪声的406名坦克兵、626名炮兵共计1032例进行听力损失调查,收集军事噪声易感者(易感组)82例,耐受者(耐受组)40例,共计122例。分别采集外周静脉血标本,从白细胞中提取DNA,PCR扩增线粒体DNA及非综合征型耳聋相关的热点突变部分片段,进行基因测序比对分析,比较两组中存在碱基改变的位点分布例数。对测序结果中存在与耳聋相关的碱基改变的个体做进一步的听力学分析。结果军事噪声易感者和耐受者的线粒体基因存在一定差异,线粒体COII基因T7684C和G7853A两个位点的变化只存在于易感组,两组间有显著性差异(P<0.05)。A827G、T961insC(异质)、T1005C、T1095C、G7444A等可能与耳聋相关的线粒体基因改变多见于易感组,耐受组也有分布。结论携带线粒体COII基因T7684C和G7853A碱基变化的个体可能对军事噪声更加易感。     

Sequence analysis of mitochondrial DNA hypervariable region I in Thai individuals

Nucleotide sequence analysis of hypervariable region I (HVRI) in human mitochondrial DNA (mtDNA) was investigated in 100 unrelated Thai individuals. A total of 85 variable sites and 423 base substitutions, which consisted of 390 nucleotide transitions and 33 nucleotide transversions were found. The following nucleotide substitutions were found: 48% at 16,223, 31% at 16,304, 30% at 16,332, and 26% at 16,129, respectively. Transition from T to C (43.7%) was the most frequent substitution. The nucleotide insertions were found at two sites with T at position 16,188 and C at position 16,194. Eighty-two haplotypes were investigated of which 72 haplotypes were unique. The most frequent haplotypes (16,108T-16,129A-16,162G-16,172C-16,304C and 16,260T-16,298C-16,355T-16,362C) were observed. From position 16,180 to 16,193, thirteen patterns of polycytosine or C-stretch were observed, whereas 68 Thai individuals were found to be similar to the references. The genetic diversity, random match probability, and discrimination power were estimated to be 0.9943, 0.0156, and 0.9844, respectively.     

DHPLC在X-连锁肾上腺脑白质营养不良分子诊断中的应用(附12例报告)

目的探讨DHPLC在X-连锁肾上腺脑白质营养不良(X-ALD)分子诊断中的应用。方法提取12个X-ALD家系及成员的外周血基因组DNA,分15个片段扩增ABCD1基因的10个外显子,应用DHPLC技术对其进行突变筛查,并对出现异常洗脱峰的PCR产物进行DNA序列测定,证实突变位点的存在。结果12个X-ALD家系存在12种不同的ABCD1基因突变,包括8个错义突变、2个移码突变和2个无义突变,即P534R、G343V、R259W、A141T、R401Q、K276E、Y174C、A314P、fs E471、fs A247、S108X和Q177X。结论DHPLC筛查结合DNA序列测定能快速有效检测出ABCD1基因突变。不同的X-ALD家系有不同的ABCD1基因突变位点,突变类型和表型之间无特殊相关关系。     

人胰岛素样生长因子-I基因P1启动子区域单核苷酸多态性-705T>C和-603T>A对启动子活性的影响(英文)

目的探讨人胰岛素样生长因子-I(IGF-I)基因P1启动子区域单核苷酸多态性(SNP)-705T>C和-603T>A对启动子活性的影响。方法招募152名健康志愿者参与本研究,取静脉血样品,使用基因组DNA提取试剂盒从血样中提取全基因组DNA。通过限制片段长度多态性分析(RFLP),对每名志愿者的SNP-705T>C和-603T>A进行基因分型,统计各基因型的频率。使用PHASE v2.1软件程序分析P1启动子单体型的类型和频率。通过荧光素酶报告基因测定不同P1启动子单体型的活力差异。结果成功提取了每名志愿者的全基因组DNA并测定了SNP-705T>C和-603T>A的基因型。基因型-705T/T、705T/C、-705C/C的频率分别与-603T/T、-603T/A、-603A/A相同,分别为43.4%、45.4%、11.2%。等位基因-705T、-705C的频率分别与-603T、-603A相同,分别为66.1%、33.9%。SNP-705T>C和-603T>A之间存在完全的连锁不平衡,其组成的P1启动子单体型只有-705T/-603T和-705C/-603A两种,频率分别为66.1%和33.9%。报告基因分析结果表明,P1启动子-705C/-603A单体型的活力显著高于-705T/-603T单体型。结论人IGF-I基因P1启动子区域SNP-705T/C和-603T/A能够影响P1启动子的活性。     

乙型肝炎病毒前C/C区及其调控基因变异的研究

目的研究慢性乙型肝炎病人中HBVpreC/C基因及其调控序列的变异和特点。方法对42例慢性乙型肝炎病人中扩增的HBVDNA各3个克隆进行序列分析。结果42例病人,HBVC基因调控序列发生T1762A1764双突变者20例,其中HBeAg阳性者7例,HBeAg阴性者13例(P<005);22例发生T1673G1799双突变。前C变异中,18例发生A1896变异,其中HBeAg阳性者6例,HBeAg阴性者12例(P<0.05)。C区变异中,AA5、AA38、AA60、AA87、AA97、AA130、AA135都是变异的热点。前C/C区还存在有插入、缺失等不同变异。结论慢性乙型肝炎病人的HBVC基因及其调控序列变异具有多样性及复杂性,与体内的免疫清除与病毒逃避免疫攻击相关,从而容易导致疾病的慢性化。     

β-防御素-1基因多态性与重症脓毒症真菌易感性的相关性研究

目的 通过病例-对照关联研究,探讨β-防御素1(DEFB1)基因多态性与重症脓毒症患者真菌感染易感性的相关性.方法 依据1992年和2002年美国胸科医师协会/美国危重病学会(ACCM/SCCM)制订的重症脓毒症诊断标准,将ICU中符合重症脓毒症诊断标准的211例患者纳入研究,并根据在ICU期间是否发生真菌感染将其进一步分为真菌感染组和对照组.通过DNA直接测序、聚合酶联反应双引物等位基因特异性扩增法(PCR-ASA)或Taqman方法检测DEFB1基因-1816A/G、-390A/T、-52A/G、-44C/G、-20A/G等5位点在两组患者中的等位基因和基因型,用遗传分析法计算其单倍型的分布频率,采用x2检验或Fisher精确概率法分析这些遗传变异与重症脓毒症患者真菌感染易感性的相关性,并以相对风险度(odds ratio,OR)反映该遗传因素与其关联的程度. 结果真菌感染组共纳入80例患者,对照组为131例.真菌感染组中男性43例(53.8%),年龄(60.81±18.30)岁;对照组男性80例(6J.1%),年龄(60.42±17.03)岁,两组间性别组成、平均年龄差异无统计学意义(P＞0.05).DEFB1基因-1816A/G、-390A/T、-52A/G、-44C/G和-20A/G位点在两组人群中都遵守Hardy-Weinberg平衡,其基因型分布和等位基因频率在真菌感染组和对照组之间差异均无统计学意义(P＞0.05).上述5个位点的常见单倍型AAACG、ATGCA、GTGGG和ATACG在真菌感染组和对照组之间的分布频率差异均无统计学意义(P＞0.05). 结论 DEFB1基因-1816A/G、-390A/T、-52A/G、-44C/G和-20A/G位点与重症脓毒症患者真菌感染的发生不相关,提示DEFB1基因遗传变异可能不是重症脓毒症患者真菌易感性的重要遗传学位点.     

重型肝炎病人的乙肝病毒前C基因变异研究

对１１例重型肝炎病人乙型肝炎病毒（ＨＢＶ）ＤＮＡ直接序列进行分析以阐述变异特点。一次或套式ＰＣＲ扩增到的ＨＢＶＤＮＡ，以不对称ＰＣＲ法制备成单链模板后直接作序列分析。结果１１个病例中，９例有１～２个氨基酸替代变异：Ｇ１８９６→Ａ的替代，产生一个终止密码，见于１例抗－ＨＢｅ阳性的病人，第１７位（ＡＡ１７）缬氨酸被苯丙氨酸替代，见于１例ＨＢｅＡｇ阴性的病人，ＡＡ１５和ＡＡ２９的丝氨酸替代变异见于２例ＨＢｅＡｇ阴性、ＨＢＶ含量甚低的病例，１８５８Ｃ→Ｔ替代见于４例病人，与ＨＢｅＡｇ表型无关，无一伴有终２８变异，呈相互排斥现象。说明重型肝炎病人的ＨＢＶ前Ｃ基因存在多种变异类型。这些变异点只见于１～２例病人，似乎不是引发重型肝炎的重要原因，Ｇ１８９６→Ａ替代变异和ＡＡ１７的苯丙氨酸替代与ＨＢｅＡｇ阴性表型相关     

Identification of newly polymorphic intron 40 markers of the von Willebrand factor gene in a Japanese population

We investigated a region of repetitive DNA located in intron 40 of the von Willebrand factor (vWF) gene (nucleotides [nt] 1639–2404; i.e., F8VWF). We identified 13 alleles and 33 genotypes in 49 unrelated Japanese individuals. The heterozygosity of the region was 0.897. Direct sequence analyses revealed five single-base substitutions, one tetranucleotide (TTAT) insertion, and seven short tandem repeats (STRs) in the intron; four of the STRs and one single-base substitution had been reported previously. The four new base substitutions we identified were 1849T>A, 2122C>T, 2180C>T, and 2192C>T. The novel TTAT tetranucleotide was inserted between nt 2057 and 2058. The three newly identified STRs were 1978(TATC)_1–2, 2193(ATCT)_5–13, and 2234(TGTA)_5–7. The five single-base substitutions and the TTAT insertion were identified only with 3′ downstream of vWA allele 14.     

Cloning and characterization of an ascidian homolog of the human 8-oxoguanine DNA glycosylase (Ogg1) that is involved in the repair of 8-oxo-7,8-dihydroguanine in DNA in Ciona intestinalis

PURPOSE: It is of interest to perform a systematic comparative analysis of the conserved domains in DNA glycosylases and the evolution of DNA base excision repair systems. Furthermore, it is important to characterize the roles and regulation of base excision repair during the development of organisms. To address these issues, we first identified 8-oxo-7,8-dihydroguanine (8-oxoG)-DNA glycosylase (Ogg1) of the ascidian Ciona intestinalis as a good model system. MATERIALS AND METHODS: A cDNA clone coding for a peptide with homology to human Ogg1 was identified in the expressed sequence tag (EST) database from the Ciona cDNA resources. We examined whether CiOgg1 has DNA glycosylase/AP (apurinic/apyrimidinic) lyase activities for 8-oxoG-containing oligonucleotide. Furthermore, the expression level of CiOgg1 was compared in various tissues of Ciona intestinalis. RESULTS: The CiOgg1gene encoded a protein of 351 amino acids, which shows 37% identity of amino acid sequence with human Ogg1. The Helix-hairpin-Helix motif was highly conserved. The ascidian enzyme had functional 8-oxoG-DNA glycosylase/AP lyase activities, which removed 8-oxoG opposite cytosine from DNA. Expression of the CiOgg1 significantly reduced the frequency of spontaneous G:C to T:A transversions in E. coli mutM mutY. The highest expression level was observed in testis in Ciona intestinalis. CONCLUSIONS: The structure and functions of Ogg1 are well conserved in Ciona intestinalis. CiOgg1 is involved in the repair of 8-oxoG in DNA in Ciona intestinalis.     

Mutations caused by gamma-radiation-induced double-strand breaks in a shuttle plasmid replicated in human lymphoblasts.

The mutagenicity of open-circular DNA (containing base damage and single-strand breaks) and linear DNA (containing base damage, single-strand breaks, and one double-strand break) produced in vitro by gamma-irradiation of shuttle vector pZ189, was analysed after the plasmid's repair and replication in the human lymphoblast line, GM606. By comparing the survival, mutation frequency, and types of mutations in descendants from the two DNA forms, the effects of the double-strand break were determined. The percentage of viable plasmids from linear DNA was two-fold lower than that from open-circular DNA, 7.8 versus 14.0 (compared with unirradiated, control DNA). The mutation frequency in progenies of the open-circular plasmid was 4.2 +/- 1.7 x 10(-3), compared with 7.8 +/- 0.1 x 10(-3) in progenies of the linear DNA, again, nearly a two-fold difference. Approximately 59% of the mutations from the linear DNA were deletions and 34% were base substitutions. In contrast, only 13% of mutations from open-circular DNA were deletions, but 87% were base substitutions. All recoverable deletions were small, ranging from 1 to 205 base pairs, and the majority contained direct repeats at the deletion junctions, indicating non-homologous recombinations. Thus, mutations found among descendants from the linear and open-circular DNAs were qualitatively similar but quantitatively different. The data suggests that producing one double-strand break in DNA by ionizing radiation causes a two-fold increase in both lethality and mutation frequency.