Experimental evidence for an alternative to directed mutation in the bgl operon.

The directed mutation hypothesis suggests that some mutations occur more often when selectively advantageous than when neutral or disadvantageous, challenging the principle that the selective value of a mutation does not affect the rate of its occurrence. Mutations in the bgl operon of Escherichia coli have been reported to be a case of directed mutation. E. coli K12 strains chi342LD cannot grow on salicin but derivatives with two mutations in the bgl operon, an excision of IS150 (formally called IS103) from bglF and a point mutation or insertion in bflR, grow rapidly on this sugar. When chi342LD is grown on a medium containing salicin, bglF excision mutants accumulate to a frequency of greater than 1%, even though these mutants are reportedly unable to grown on salicin, and Sal+ double mutants subsequently attain a high frequency. Comparable accumulations of excision mutants and Sal+ double mutants are not observed in the absence of salicin. As salicin is not mutagenic, it has been suggested that excision mutations in bglF might serve only to create the potential for a secondary selectively advantageous mutation. We show here, however, that these double mutants can be accounted for by spontaneous mutation to intermediate genotypes in non-growing populations, coupled with slow growth of some of these intermediates on salicin, which enables their populations to reach a size where secondary mutations allowing rapid growth on salicin become common.     

酿酒酵母的连续复合诱变及其突变株的快速筛选

为了获得高产的工业用酿酒酵母(Saccharomyces cerevisiae)菌株,利用己烯雌酚(DES)和UV对酿酒酵母菌QD进行连续复合诱变,采用改进的发酵小管集气法结合乙醇定量测定来进行突变株的筛选,并与常规复合诱变及筛选方法进行比较.结果表明:连续复合诱变的正向突变率最大值为18.9%,与常规诱变方法的最大正向突变率15.6%相比提高了21.2%,说明运用该诱变方法能获得较高的正向突变率;改进的筛选方法可以快速筛选出目的突变株,同时,获得了一株高产乙醇的突变株,在甘蔗汁发酵培养基中其乙醇产量达到9 720 mg/100 mL,比常规诱变方法筛选到的突变株乙醇产量(9 170 mg/100 mL)高出6.0%;与常规诱变方法相比,连续复合诱变方法不仅能够得到较高的正向突变率,而且能够获得更加高产的突变株.     

一种优良半矮秆水稻品种的分离与分子鉴定

株1S是中国南方稻区杂交水稻育种广泛应用的优良籼型温敏雄性核不育系.为了改良其农艺性状,我们采用体细胞无性系诱变方法筛选半矮秆突变体.本文报道了2个体细胞无性系突变体SV1S和SV14S的表型和初步分子鉴定结果.与亲本株1S相比,突变体的高度降低,基部节间长度缩短,节间壁显著增厚,但是上部的节间变化不明显.更重要的是,我们发现赤霉素生物合成途径中的关键酶基因GA20ox-2出现了约200bp的缺失突变,导致该基因在半矮秆突变体中的转录水平降低.研究结果表明,突变体SV1S和SV14S很可能是由于同一缺失突变导致赤霉素合成受阻,从而部分导致了半矮秆性状的出现.然而,2个突变体苗期的高度相同,以及糊粉层细胞!-淀粉酶活性和赤霉素敏感性降低等性状与以前报道的sd-1突变体有所不同.因此,我们推测在SV突变体中可能还存在第二个突变位点.该结果也进一步证实了体细胞无性系诱变在水稻育种中具有良好的应用前景.     

Drastic change in idiotypic but not antigen-binding specificity of an antibody by a single amino-acid substitution

In proliferating B lymphocytes, somatic mutation of rearranged antibody variable (V)-region genes occurs at high frequency and may have a key role in the selection of these cells. It is of interest in this context to learn in which way single mutations can affect antigen binding and/or idiotypic specificity of an antibody. Previous investigations have analysed spontaneous mutants of myeloma and hybridoma cells in which the mutation affected the antigen-binding specificity of the antibody. Here we describe an antibody mutant that has fully retained antigen-binding specificity but has lost or drastically changed all V-region antigenic determinants (idiotopes) of the wild type as defined by monoclonal anti-idiotope antibodies. The mutant phenotype is generated by a glycine to arginine exchange in the middle of the diversity (D) element, at position 103 of the heavy chain.     

Complete mutagenesis of the HIV-1 protease

Retroviruses encode a protease which needs to be active for the production of infectious virions. A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease. Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein. Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation. These results show that random mutagenesis can be used to identify functionally important regions within a protein. Mutants with conditional phenotypes have also been identified within this collection.     

Random components in mutagenesis

The mutability of DNA varies enormously from one base pair to another. Part of this variation is due to the specificity of the reaction between mutagens and base, but much of the variation is due to unknown causes. A genetic system developed by Miller and colleagues allows the mutation frequencies of a large number of different base pairs in the lacI gene of Escherichia coli to be compared. For example, Coulondre and Miller found that the sites most readily mutated by UV light are almost 100 times more often mutated than the least susceptible sites. A recently completed study of mutagenesis with neocarzinostatin (NCS) in the lacI gene has prompted us to re-examine some previous studies, of mutagenesis in this gene. Our analysis, reported here, suggests that the mutations induced by certain mutagens fall into two classes: mutations in one class are clearly distributed non-randomly, that is, they are very common at some sites and significantly less common at others; mutations in the second class, however, occur at low frequency and appear to be randomly distributed. Both classes of mutations seem to occur only at damaged bases.     

大肠杆菌ATCase抗反馈抑制突变体的构建及其对胞苷积累的影响

为解决胞苷生物合成途径中天冬氨酸氨甲酰转移酶受胞苷三磷酸反馈抑制调节的问题,通过对其碱基序列和蛋白质结构分析,利用基因定点突变的方法构建了大肠杆菌的ATCase突变酶,得到三个突变体:M1(H20L)、M2(K60E)、M3(K94E),并在E.coli DH5α中对融合蛋白进行了表达.酶活测定表明,M1、M2、M3的ATCase酶相对活性都比野生型M0的高,分别为野生型M0的1.10、1.22和1.37倍,且比活力都有不同程度提高.与含野生型pyrBI基因的M0相比,含突变型基因的M1、M2和M3均对15,mmol/L的CTP具有强的抗反馈抑制作用,且M1、M2和M3的抗CTP反馈抑制作用分别是M0的5.4、6.0和8.5倍.最后将各突变质粒转入到E.coli Cyt10(Δcdd)中进行发酵培养,结果表明,与未含突变基因菌株相比,各含突变基因菌株的胞苷积累量均有不同程度的提高,说明ATCase定点突变使胞苷的合成积累途径得到了不同程度的强化.     

Germline mosaicism and Duchenne muscular dystrophy mutations

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disease with an incidence of approximately 1 in 3,500 newborn boys. The DMD locus has a high mutation frequency: one third of the cases is thought to result from a new mutation. Linkage studies using probes to detect restriction fragment length polymorphisms and DNA deletion studies have greatly improved DMD carrier detection and prenatal diagnosis. Here we report on two families in which a pERT87 (DXS164) deletion was transmitted to more than one offspring by women who showed no evidence for the mutation in their own somatic (white blood) cells. We also show that the deletion in both siblings in one of the families is identical, indicating that the deletion must have occurred during mitosis in early germline proliferation, leading to a germline mosaicism. This phenomenon may turn out to be a major factor contributing to the induction of DMD mutations, and has important implications for the counselling of DMD families.     

Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population

An RNA virus population does not consist of a single genotype; rather, it is an ensemble of related sequences, termed quasispecies. Quasispecies arise from rapid genomic evolution powered by the high mutation rate of RNA viral replication. Although a high mutation rate is dangerous for a virus because it results in nonviable individuals, it has been hypothesized that high mutation rates create a 'cloud' of potentially beneficial mutations at the population level, which afford the viral quasispecies a greater probability to evolve and adapt to new environments and challenges during infection. Mathematical models predict that viral quasispecies are not simply a collection of diverse mutants but a group of interactive variants, which together contribute to the characteristics of the population. According to this view, viral populations, rather than individual variants, are the target of evolutionary selection. Here we test this hypothesis by examining the consequences of limiting genomic diversity on viral populations. We find that poliovirus carrying a high-fidelity polymerase replicates at wild-type levels but generates less genomic diversity and is unable to adapt to adverse growth conditions. In infected animals, the reduced viral diversity leads to loss of neurotropism and an attenuated pathogenic phenotype. Notably, using chemical mutagenesis to expand quasispecies diversity of the high-fidelity virus before infection restores neurotropism and pathogenesis. Analysis of viruses isolated from brain provides direct evidence for complementation between members in the quasispecies, indicating that selection indeed occurs at the population level rather than on individual variants. Our study provides direct evidence for a fundamental prediction of the quasispecies theory and establishes a link between mutation rate, population dynamics and pathogenesis.     

Frequent mtDNA mutations and its role in gastric carcinogenesis

In order to disclose the relationship between mutations of mitochondrial DNA (mtDNA) and gastric carcinogenesis, we screened the entire mtDNA sequence in 30 cases of human gastric cancer and matched normal tissues by using denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. Our data showed that high frequency (66.7%, 20/30) of mitochondrial genome mutation occurred in gastric cancer. Among these variants, 17 cases (56.7%, 17/30) were identified to be somatic mutation. High level mutant frequency was found in ND4, ND5 coding genes and D-loop control region, which was 36.7%, 26.7% and 30% respectively. Comparing with complexes Ⅲ, Ⅳ and Ⅴof the electron transport chain, we found that variants appeared to be more frequent in the subunit genes of complexⅠ. Most of mutations were base substitutions (85.4%, 41/48). Our results suggested that mutations of subunit genes encoding complexⅠ, especially ND3, ND4 and ND5 genes, might contribute to human gastric carcinogenesis.     

A large increase in enzyme-substrate affinity by protein engineering

A single point mutation has been engineered in the tyrosyl-tRNA synthetase that improves its affinity (KM) for its substrate ATP by a factor of 100. In the crystal structure of the tyrosyl tRNA synthetase (of Bacillus stearothermophilus), the side-chain hydroxyl of Thr 51 appears to make a weak hydrogen bond with the AMP moiety of the substrate intermediate, tyrosyl adenylate. In the absence of substrate, however, the hydroxyl group should make a strong hydrogen bond with water which would favour dissociation of the enzyme-substrate complex. We have used oligodeoxynucleotide-directed mutagenesis to construct two point mutants at this site: one to remove the hydroxyl group (Thr 51 leads to Ala 51) and the other, in addition, to distort the local polypeptide backbone (Thr 51 leads to Pro 51). We report here that both mutants have increased activity (kcat/KM for ATP) but one mutant (Pro 51) shows a massive 25-fold increase due mainly to a lowered KM for ATP. This demonstrates dramatically the potential of in vitro mutagenesis for improving the affinity of an enzyme for its substrate.     

High mutation rate and predominance of insertions in the Caenorhabditis elegans nuclear genome

Mutations have pivotal functions in the onset of genetic diseases and are the fundamental substrate for evolution. However, present estimates of the spontaneous mutation rate and spectrum are derived from indirect and biased measurements. For instance, mutation rate estimates for Caenorhabditis elegans are extrapolated from observations on a few genetic loci with visible phenotypes and vary over an order of magnitude. Alternative approaches in mammals, relying on phylogenetic comparisons of pseudogene loci and fourfold degenerate codon positions, suffer from uncertainties in the actual number of generations separating the compared species and the inability to exclude biases associated with natural selection. Here we provide a direct and unbiased estimate of the nuclear mutation rate and its molecular spectrum with a set of C. elegans mutation-accumulation lines that reveal a mutation rate about tenfold higher than previous indirect estimates and an excess of insertions over deletions. Because deletions dominate patterns of C. elegans pseudogene variation, our observations indicate that natural selection might be significant in promoting small genome size, and challenge the prevalent assumption that pseudogene divergence accurately reflects the spontaneous mutation spectrum.     

He-Ne激光诱变选育产氢拜氏梭菌

采用He-Ne激光器对拜氏梭菌(Clostridium beijerinckii)进行激光诱变育种,对激光诱变参数进行优化,结果表明,随着照射剂量的变化,存活率随之变化,输出功率和辐射时间均由正突变率决定。在最佳照射条件(19 m W,16 min)下,得到稳定的阳性突变株BH4。在初始葡萄糖浓度为10 g/L的分批培养中,突变株每克葡萄糖的最终产氢量为260. 7 m L,比野生菌高出28. 9%。酶测定显示,诱变后氢化酶活性增加,这可能有助于提高产氢量。此外,研究了分批培养物中突变体BH4及其野生菌的动力学,其初始葡萄糖浓度为1～10 g/L。动力学参数还表明,突变菌比其野生菌具有更强的产氢能力。说明该He-Ne激光诱变育种技术可以在产氢微生物领域中应用。     

GCC box in<Emphasis Type="Italic">Arabidopsis PDF1.2</Emphasis> promoter is an essential and sufficient cis-acting element in response to MeJA treatment

The expression of Arabidopsis PDF1.2 gene isregulated by jasmonic acid (JA) and ethylene (ET). It also has been well documented that GCC box is an element responsive to ET, however, the responsive mechanism of JA in such plant defense gene expression is unclear. In this paper, the authors define the essential cis-acting element in PDF1.2 promoter responsive to methyl jasmonate (MeJA) through fragment deletions and site-directed mutageneses combiningAgrobacterium-mediated transient reporter gene expression in tobacco leaves. Firstly, the MeJA inducible expression o fPDF1.2 was confirmed by using the upstream -1.86 kb fragment of PDFI.2 gene. Secondly, the upstream -300— -243 bp fragment of the promoter was evidenced to respond to MeJA. To further characterize this promoter region, three point mutations were introduced into the -300— -243 bp fragment of the promoter. This result showed that the mutation of GCC box abolished MeJA induction, whereas the mutations of the G box-like and the imperfect palindrome sequence did not significantly decrease MeJA inducible effect, indicating that GCC box in PDFI.2 is essential for MeJA induction. The sufficient responsiveness to MeJA of this GCC box was further investigated by 4xGCC fused upstream to the CaMV 35S minimal promoter. This result suggested that the fused promoter was able to activate reporter gene expression in response to MeJA. Thus these results indicate that the GCC box in PDFI.2 is an essential and sufficient element to confer MeJA induction.     

腺苷生产菌株的回复突变和性质检验

腺苷具有重要的医学价值,由RNA降解获取腺苷成本极高,用微生物发酵法生产腺苷具有重大的经济意义.作为腺苷的生产菌株,黄嘌呤缺陷型的枯草芽孢杆菌如果在生产过程中发生回复突变将会降低腺苷产量.本实验研究了枯草杆菌的黄嘌呤缺陷性菌株是否会回复突变、以及突变菌株的各种性质,进行了V.P.,硝酸盐,高盐浓度,淀粉水解检验,以及进一步的IMP脱氢酶、黄嘌呤氧化酶、腺苷脱氨酶的活性分析,发现大量菌株发生回复突变,该缺陷型菌株遗传非常不稳定,这些突变菌株与生产菌株相比,IMP脱氢酶活发生显著变化,黄嘌呤氧化酶与腺苷脱氨酶的活力没有明显变化.     

利用空间诱变选育辅酶Q10高产菌

 以空间搭载为诱变手段,筛选可用于工业化生产的辅酶Q10高产菌株。以类球红细菌（Rhodobacter sphaeroides）为出发菌株进行空间搭载,根据菌落形态进行初筛,摇瓶发酵进行复筛,HPLC检测含量等方法筛选辅酶Q10高产菌株。最终得到名为Shenzhou6的突变株产量比对照菌株提高30％,产量可达（0.8+0.02）g/L,经过优化培养可满足工业生产需要。同时也说明空间诱变因其复杂的空间环境条件,可以作为一种诱变手段进行工业化菌样的筛选。     

Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene

The chief limitation of phenotype-based genetic screening in mammalian systems is the diploid nature of the genome. Cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of loss of heterozygosity. Here we have used a tetracycline-regulated Blm allele (Blm(tet)) to introduce bi-allelic mutations across the genome in mouse embryonic stem (ES) cells. Transient loss of Blm expression induces homologous recombination not only between sister chromatids but also between homologous chromosomes. We considered that the phenotype of ES cells bearing bi-allelic mutations would be maintained after withdrawal of the tetracycline analogue doxycycline. Indeed, a combination of N-ethyl-N-nitrosourea mutagenesis and transient loss of Blm expression enabled us to generate an ES cell library with genome-wide bi-allelic mutations. The library was evaluated by screening for mutants of glycosylphosphatidylinositol-anchor biosynthesis, which involves at least 23 genes distributed throughout the genome. Mutants derived from 12 different genes were obtained and two unknown mutants were simultaneously isolated. Our results indicate that phenotype-based genetic screening with Blm(tet) is very efficient and raises possibilities for identifying gene functions in ES cells.     

人囊性纤维化跨膜电导调节子蛋白的变异模式研究

【目的】编码囊性纤维化跨膜电导调节子（Cystic fibrosis transmembrane conductance regulator，CFTR）蛋白的基因突变可引起囊性纤维化，但该蛋白错义点突变的变异模式尚无报道。【方法】先用氨基酸对可预测性为指标将人CFTR蛋白及其178个错义点突变的氨基酸序列转换成标量序列，然后分析变异前后被替换掉的和替换出的氨基酸对的变化。【结果】97．19％的变异发生在不可预测的氨基酸对；87．08％的变异涉及1个或2个被替换掉的氨基酸对，其实际频率大干预测频率；15．17％的变异带来1个或2个替换出的氨基酸对，它们在正常的CFTR蛋白是不存在的；共有122个变异导致替换出的氨基酸对的实际频率小干预测频率。【结论】不可预测的氨基酸对对变异更敏感，变异的趋势是缩小氨基酸对实际频率和预测频率之间的差距，使氨基酸对的构成更加随机化，而人CFTR蛋白的这种退行性变导致了囊性纤维化。