低能N+辐照拟南芥诱导基因组DNA碱基变异分析

用低能N+离子束注入拟南芥后获得的稳定突变体T80Ⅱ作为实验材料, 对突变体植株进行了RAPD标记, 并将T80Ⅱ和对照部分RAPD特异条带进行克隆测序和DNA序列分析. 结果显示, 在可分辨的总计397个RAPD条带中, T80Ⅱ株系中有52个条带表现出差异, 包括条带的缺失和增加, 条带变异率为13.1%; 克隆的T80Ⅱ序列中, 平均每16.8个碱基出现1个碱基变异位点, 表现出较高频率的碱基突变. 碱基突变类型包括碱基的颠换、转换、缺失、插入等. 在检测到的275个碱基突变中, 主要是单碱基置换(97.09%), 碱基缺失或者插入的比例较小(2.91%). 在碱基置换中, 转换的频率(66.55%)高于颠换的频率(30.55%). 此外, 构成DNA的4种碱基均可以被离子束辐照诱发变异, 而且每一种碱基都可以被其他3种碱基所替换, 但是胸腺嘧啶(T)的辐射敏感性要高于其他3种碱基. 通过分析突变碱基周边序列, 对低能N+离子注入拟南芥突变体引发的碱基突变热点进行了讨论.     

低能N+辐照拟南芥诱导基因组DNA碱基变异分析

用低能N~+离子束注入拟南芥后获得的稳定突变体T80Ⅱ作为实验材料,对突变体植株进行了RAPD标记,并将T80Ⅱ和对照部分RAPD特异条带进行克隆测序和DNA序列分析。结果显示,在可分辨的总计397个RAPD条带中,T80Ⅱ株系中有52个条带表现出差异,包括条带的缺失和增加,条带变异率为13.1％;克隆的T80Ⅱ序列中,平均每16.8个碱基出现1个碱基变异位点,表现出较高频率的碱基突变。碱基突变类型包括碱基的颠换、转换、缺失、插入等。在检测到的275个碱基突变中,主要是单碱基置换(97.09％),碱基缺失或者插入的比例较小(2.91％)。在碱基置换中,转换的频率(66.55％)高于颠换的频率(30.55％)。此外,构成DNA的4种碱基均可以被离子束辐照诱发变异,而且每一种碱基都可以被其他3种碱基所替换,但是胸腺嘧啶(T)的辐射敏感性要高于其他3种碱基。通过分析突变碱基周边序列,对低能N~+离子注入拟南芥突变体引发的碱基突变热点进行了讨论。     

氟氏链霉菌离子束注入突变谱的分析

用低能N⁺离子束注入转谷氨酰胺酶产生菌氟氏链霉菌后,通过试验,初步确定了注入的效应曲线,获得了一系列突变菌株。提取原始菌株和突变菌株的DNA,采用PCR反应分段扩增出转谷氨酰胺酶基因进行单链构象多态性分析(SSCP),并将特异性条带克隆测序进行基因突变型的鉴定,分析离子束注入引起链霉菌基因的基因突变类型及特点。结果显示:碱基变异的类型包括转换、颠换和缺失。在检测到的24个碱基突变中,主要是碱基的置换(87.5%),碱基缺失的比例比较小(12.5%)。在碱基置换中,转换的频率(58.3%)高于颠换的频率(29.2%)。转换主要以C→T,A→G为主,颠换以G→T,C→G为主。此外构成DNA的4种碱基均可以被离子束辐照诱发变异,其中胞嘧啶发生突变的频率较高。     

大肠杆菌离子束诱变及rpoB基因两个新的利福平抗性位点鉴定

以60Co-γ射线辐照为参照体系,研究了低能氮离子诱发大肠杆菌利福平抗性突变。结果表明,低能氮离子注入具有损伤轻而突变率较高的特点。碱基置换型突变与其检出频率分析表明,CG→TA、GC→AT、AT→GC转换与AT→TA颠换为低能氮离子诱发大肠杆菌活体细胞内的高频突变,占检出总突变数的875% (77/88)。并鉴定出大肠杆菌rpoB基因中两个新的利福平抗性决定位点。位点一位于1551位鸟嘌呤脱氧核苷酸（dG）被胞嘧啶脱氧核苷酸（dC）取代,导致Gln517 (谷氨酰胺残基) 被His (组氨酸) 替代;位点二位于1692的胞嘧啶脱氧核苷酸（dC）被胸腺嘧啶脱氧核苷酸（dT）替代,导致Pro564 (脯氨酸残基) 被Leu (亮氨酸) 取代,使突变子产生抗性。其中位点一还未见报道,位点二的同义突变已有报道,但1692位点C到T的核苷酸突变并没有得到鉴定。     

PCR—SSCP与测序技术相结合检测小麦耐盐突变体

根据位于小麦第四同源群上与耐盐有关的gf-2.8基因的编码区序列设计1对引物,分别以两个耐盐突变体及其亲本的总DNA为模板进行PCR扩增,在5个供试材料中均扩增出1条约685bp的目的条带,SSCP电泳显示突变体974915与其他供试材料之间存在差异。测序表明冀麦24和其耐盐突变体8901-17的扩增产物序列与gf-2.8基因的发表序列相同,这表明突变体8901-17的突变位点不在该基因上,而另一耐盐突变体974915的序列中则至少存在2个单碱基突变,有一处突变导致了氨基酸的变化,该突变位点位于gf-2.8基因的保守区域内。     

应用RAPD技术辅助蝴蝶兰辐射育种

应用RAPD技术对蝴蝶兰辐射诱变苗进行分析。结果表明,对蝴蝶兰5种不同形态的诱变苗和对照苗进行PCR扩增,13个引物所产生的RAPD谱带大小为200~3 000 bp,共扩增77条带,多态性带61条,多态率79.22%。13个引物平均扩增的带数为5.92,引物S0036最多,为9条带。比较诱变苗与对照苗谱带,两者有差异,表明诱变苗是由基因突变引起。     

用DREAM技术纠正人工合成基因中的突变

目的：介绍一种简便、有效的定点突变技术。方法：根据突变位点附近的DNA序列推导出氨基酸序列,再以此氨基酸序列进行逆翻译,这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体（silent mutants）,这些突变体中包含大量的限制性内切酶位点,选择合适的酶切位点设计引物用PCR技术扩增两侧DNA片段,然后以相应酶切融合这两个片段即可完成定点突变。结果：用该方法成功地在人工合成的含有缺失的可溶性组织因子基因的472位插入C,T两个碱基,校正了阅读框架,获得了预期的目的基因。结论：该方法简便、有效, 避免了多轮PCR和合成长引物导致突变的可能性,这种改进的PCR 定点诱变技术我们称之为“设计限制酶辅助突变”（Designed Restriction Enzyme Assisted Mutagenesis, DREAM）。此技术简单方便, 诱变的成功率高, 适于实验室常规应用。     

γ射线诱导质粒pGEM-3Zf(-)DNA lacZ基因突变的序列分析

利用质粒ｐＧＥＭ－３Ｚｆ（－）ＤＮＡ上带有的ｌａｃＺ基因,通过Ｘ－ｇａｌ显色平板筛选经＾６０Ｃｏγ辐射诱导形成的突变体。对４个突变体的ｌａｃＺ基因进行序列分析发现,所有突变体无一例外地检测到碱基变化。在被测序的４８９ｂｐ范围内,一个突变体发生的碱基变异达２－７个位点。碱基变异的类型包括颠换（５７％）、转换（１９％）、缺失（１９％）及插入（５％）。在碱基置换中,以Ｃ／Ｇ→Ａ／Ｔ颠换最多（占５０％）。     

65份野生油茶种质遗传多样性的ISSR和RAPD标记分析

利用ISSR和RAPD标记,对名邛台地野生油茶种质进行遗传多样性分析。从60条简单重复序列引物中筛选出16条引物,在65份样品中共扩增出213条带,其中多态位点为203个,多态位点百分率为95.31%;从30条寡居核苷酸引物中筛选出8条引物,共扩增出105条带,其中多态性位点94个,多态位点百分率为89.52%。结果表明:名邛台地野生油茶种质具有较丰富的遗传多样性,ISSR和RAPD标记可以应用于油茶种质遗传多样性分析。     

氮离子诱变筛选内切葡聚糖酶高产菌株及其基因突变研究

目的：离子注入枯草芽孢杆菌筛选高产内切葡聚糖酶突变菌株,同时进行其酶活性研究,并克隆该基因,研究离子注入对其诱变效应。方法：低能氮离子重复注入枯草芽孢杆菌,筛选获得1株高产内切葡聚糖酶突变菌株Bac11。DNS法测定酶活性。PCR扩增获得出发菌株Bac01和突变菌株Bac11内切葡聚糖酶基因,并对核酸序列及预测氨基酸序列进行多重比对。结果：突变菌株Bac11内切葡聚糖酶活性从93.33IU提高到381.89IU。多重比对Bac01和Bac11内切葡聚糖酶基因编码区1500bp序列,当中有10个碱基发生突变,预测氨基酸序列中有5个氨基酸残基发生变化,且都在其基因纤维素结合域部分。结论：低能氮离子重复注入对枯草芽孢杆菌内切葡聚糖酶活性及其基因有明显的诱变累加效应。     

Changes in DNA base sequences in the mutant of Arabidopsis thaliana induced by low-energy N+ implantation

Arabidopsis thaliana (L.) Heynh has many advantages for genome analysis, including a short generation time, small size, large number of offspring, and a relatively small nuclear genome in comparison to other angiosperms and contains a low proportion of repetitive DNA comparatively. Furthermore, the analysis of the completed genome sequence of A. thaliana has been reported[1]. Low-energy ion implantation has attracted more and more attention from researchers in China and Japan since recent s…     

Changes in DNA base sequences in the mutant of Arabidopsis thaliana induced by low-energy N+ implantation

To reveal the mutation effect of low-energy ion implantation on Arabidopsis thaliana in vivo, T80II, a stable dwarf mutant, derived from the seeds irradiated by 30 keV N⁺ with the dose of 80×10¹⁵ ions/cm² was used for Random Amplified Polymorphic DNA (RAPD) and base sequence analysis. The results indicated that among total 397 RAPD bands observed, 52 bands in T80II were different from those of wild type showing a variation frequency 13.1%. In comparison with the sequences of A. thalianain GenBank, the RAPD fragments in T80II were changed greatly in base sequences with an average rate of one base change per 16.8 bases. The types of base changes included base transition, transversion, deletion and insertion. Among the 275 base changes detected, single base substitutions (97.09%) occurred more frequently than base deletions and insertions (2.91%). And the frequency of base transitions (66.55%) was higher than that of base transversions (30.55%). Adenine, thymine, guanine or cytosine could be replaced by any of other three bases in cloned DNA fragments in T80II. It seems that thymine was more sensitive to the irradiation than other bases. The flanking sequences of the base changes in RAPD fragments in T80II were analyzed and the mutational “hotspot” induced by low-energy ion implantation was discussed.     

Changes in DNA base sequences in the mutant of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> induced by low-energy N<Superscript>+</Superscript> implantation

To reveal the mutation effect of low-energy ion implantation on Ambidopsis thaliana in vivo, T80II, a stable dwarf mutant, derived from the seeds irradiated by 30 keV N⁺ with the dose of 80 X 10¹⁵ ions/cm² was used for Random Amplified Polymorphic DNA (RAPD) and base sequence analysis. The results indicated that among total 397 RAPD bands observed, 52 bands in T80II were different from those of wild type showing a variation frequency 13.1%. In comparison with the sequences of A. thaliana in GenBank, the RAPD fragments in T80II were changed greatly in base sequences with an average rate of one base change per 16.8 bases. The types of base changes included base transition, transversion, deletion and insertion. Among the 275 base changes detected, single base substitutions (97.09%) occurred more frequently than base deletions and insertions (2.91%). And the frequency of base transitions (66.55%) was higher than that of base transversions (30.55%). Adenine, thymine, guanine or cytosine could be replaced by any of other three bases in cloned DNA fragments in T80II. It seems that thymine was more sensitive to the irradiation than other bases. The flanking sequences of the base changes in RAPD fragments in T80II were analyzed and the mutational “hotspot” induced by low-energy ion implantation was discussed.     

Sequence analysis of lacZ- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ^- mutation frequency at 20% survival could reach (3.6–16.8) × 10⁴, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ^-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined); this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G. The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily substituted.     

Sequence analysis of lacZ- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ^- mutation frequency at 20% survival could reach (3.6–16.8) × 10⁴, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ^-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined); this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G. The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily substituted.     

A Novel Role for Escherichia coli Endonuclease VIII in Prevention of Spontaneous G→T Transversions

In the bacterium Escherichia coli, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the nth and nei genes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C-->A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C-->T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in fpg mutY double mutants was significantly enhanced by the addition of the nei mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C-->T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.     

Mechanism of mutation by thymine starvation in Escherichia coli: clues from mutagenic specificity.

To probe the mechanisms of mutagenesis induced by thymine starvation, we examined the mutational specificity of this treatment in strains of Escherichia coli that are wild type (Ung+) or deficient in uracil-DNA-glycosylase (Ung-). An analysis of Ung+ his-4 (ochre) revertants revealed that the majority of induced DNA base substitution events were A:T----G:C transitions. However, characterization of lacI nonsense mutations induced by thymine starvation demonstrated that G:C----A:T transitions and all four possible transversions also occurred. In addition, thymineless episodes led to reversion of the trpE9777 frameshift allele. Although the defect in uracil-DNA-glycosylase did not appear to affect the frequency of total mutations induced in lacI by thymine deprivation, the frequency of nonsense mutations was reduced by 30%, and the spectrum of nonsense mutations was altered. Furthermore, the reversion of trpE9777 was decreased by 90% in the Ung- strain. These findings demonstrate that in E. coli, thymine starvation can induce frameshift mutations and all types of base substitutions. The analysis of mutational specificity indicates that more than a single mechanism is involved in the induction of mutation by thymine depletion. We suggest that deoxyribonucleoside triphosphate pool imbalances, the removal of uracil incorporated into DNA during thymine starvation, and the induction of recA-dependent DNA repair functions all may play a role in thymineless mutagenesis.     

Influence of smoking and donor age on the spectrum of in vivo mutation at the HPRT-locus in T lymphocytes of healthy adults

Types and frequencies of in vivo mutation in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene was studied in 142 T cell mutants from 78 healthy nonsmoking and smoking adults with a mean of 65 years. The HPRT mutant frequency in the nonsmokers was 18.7±12.0×10⁻⁶, and in the smokers 26.6±18.5×10⁻⁶ (mean±S.D., P<0.01). Among 107 single base pair substitutions (SBS) in the coding region of the HPRT gene, one new mutable site, one novel nonsense mutation and three not previously reported SBS were identified. Transitions accounted for 59% of the SBS and transversions for 41%. GC>AT transitions were the predominant type of mutation, with 50% of all SBS. The mutations showed a nonrandom distribution along the coding sequence, with three significant hotspots at positions 143, 197 and 617 (13, 14 and 7 mutations, respectively). There was no difference between smokers and nonsmokers with regard to the distribution of mutations at these hotspot positions. However, 85% of the mutations at GC base pairs and 88% of the mutations at AT base pairs in smokers occurred at sites with guanine or thymine, respectively, in the nontranscribed DNA strand. Moreover, smokers had a higher frequency of transversions and lower frequency of transitions than nonsmokers did. Particularly, GC>TA transversions were increased in smokers (11%) compared to nonsmokers (2%), which suggests that tobacco-smoke induced adducts at guanine bases in the nontranscribed DNA strand contributes to the increase of HPRT mutation in smokers. Overall, these results were very similar to the mutational spectra in two younger study populations reported previously [K.J. Burkhart-Schultz, C.L. Thompson, I.M. Jones, Spectrum of somatic mutation at the hypoxanthine phosphoribosyltransferase (HPRT) gene of healthy people, Carcinogenesis 17 (1996) 1871–1883; A. Podlutsky, A.-M. Österholm, S.-M. Hou, A. Hofmaier, B. Lambert, Spectrum of point mutations in the coding region of the hypoxanthine-guanine phosphoribosyltransferase, Carcinogenesis 19 (1998) 557–566]. With the possible exception of an increase of mutations at hotspot position 143, and a decrease of 5-methylcytosine deamination mediated transitions at CpG-sites in the older individuals, there were no differences between the mutational spectra of old and young adults. In conclusion, both smoking and ageing seem to have minor influences on the spectrum of HPRT mutation in T cells.     

Similarity of Mutation Spectra of the Mitochondrial DNA Hypervariable Segment 1 in Homo and Pan Species

The mutation spectrum of mtDNA hypervariable segment 1 (HVS1) was compared for east chimpanzee Pan troglodytes schweigfurthi and human. The two HVS1 had much the same nucleotide composition, and their mutation spectra were similar in major characteristics (substantial prevalence of transitions over transversions, pyrimidine transitions over purine ones, and C T over T C). DNA strand displacement (dislocation) during replication was identified as a major mechanism of context-dependent mutagenesis in human and chimpanzee mtDNAs. Nucleotide positions with mutations fitting the model of dislocation mutagenesis accounted for 21% of all variable positions in the chimpanzee HVS1. Variable motifs proved to be similar in the chimpanzee and human HVS1. Comparison of the Neanderthal and modern human HVS1 nucleotide sequences showed that most variable nucleotides are in DNA sites allowing context-dependent mutagenesis.     

Transversion-specific purine analogue mutagens and the mechanism of hydroxylaminopurine mutagenesis

The tryptophan synthetase gene A series of mutants in E. coli has been used to examine the mutational specificity of over 80 purine base analogues. 4 purine analogues have been discovered that solely cause transversions. Evidence is presented that hydroxylaminopurine mutagenesis is caused by a covalent reaction of these compounds with DNA. The transversion-causing purine analogues are derivatives of 2-aminopurine (2AP) and 2,6-diaminopurine (2,6DAP). They stimulate the full reversion frequency of those trp A which can revert through an AT----CG transversion. 8 purine base analogues have been found that induce the AT----CG transversion at the trp A88 site; and 2-amino-6-methylaminopurine (2A6MAP) stimulates by 124-fold, 2-amino-6-ethylaminopurine by 20-fold, 2-methylaminopurine (2MAP) by 9.4-fold, 2,6-bismethylaminopurine by 25-fold, 2AP by 230-fold, 2,6DAP by 15-fold, 2.6-diaminopurine riboside by 5-fold, and 2-hydroxylaminopurine by 11-fold. The last 4 analogues also cause transitions. 2A6MAP, 2-amino-6-ethylaminopurine and 2,6-bismethylaminopurine stimulate only the AT----CG transversion while 2MAP additionally gives rise to AT----TA transversions. By testing other negative 2AP derivatives, the structural requirements necessary for AT----CG transversion mutagenesis have been determined. All 12 hydroxylaminopurine base analogues tested, 2,6-dimethoxyaminopurine and 2-hydrazinopurine were found to cause transition mutations. All of the compounds stimulated the AT----GC transition (by up to 1000-fold) and 11 of the 14 base analogues raised the GT----AT transition (by up to 450-fold). On increasing the hydroxylaminopurine concentration, the mutation frequency also increased concomitantly. Since 6-hydroxylamino-9-methylpurine and 6-methylhydroxylaminopurine cause transitions, the mechanism of hydroxylaminopurine mutagenesis cannot be entirely due to an alteration in tautomeric equilibria or "wobble" type base mispairing. It is proposed that a major mechanism for hydroxylaminopurine mutagenesis is due to the reaction of these compounds with the O6-position of guanine and the O4-position of thymine.