Effect of gene amplification on threonine production by yeast

In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the amino acid production by Saccharomyces cerevisiae. The genes used were wild-type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3-R2 and HOM3-R6), that code for feedback-insensitive aspartate kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate kinase and that, probably, a second and less important regulation takes place at the level of the homoserine kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3-R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully. (c) 1996 John Wiley & Sons, Inc.     

Investigation of mutant frequency at the HPRT locus and changes in microsatellite sequences in healthy young adults

In an attempt to understand the inter-individual variation that occurs in in vivo mutant frequency at the HPRT locus, we have examined the effect of polymorphisms in genes for metabolic enzymes on the mutation rate. In the same population of human volunteers, the background variant frequency in a number of microsatellite sequences was studied to determine individual variation in the capacity to repair mismatches in these sequences. The HPRT mutant frequency of T-cells isolated from a group of 49 healthy, non-smoking adults varied from 0.25 to 9.64×10⁻⁶. The frequency of polymorphisms in CYP1A1, GSTM1 and NAT2 among these individuals was similar to those published, and when subjected to univariate analysis these polymorphisms showed no influence on the HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator status in NAT2 (P<0.05) which was associated with higher mutant frequency. Analysis of 30 microsatellite sequences in 20 HPRT proficient clones per individual showed only six alterations in total, giving an overall mutation rate per allele of 0.01%, whilst three alterations were found in five HPRT deficient clones per individual examined for changes in 10 microsatellites, giving an overall mutation rate per allele of 0.3%. Thus, the alterations detected are probably due to background mutations and not to differences in mismatch repair capacity.     

New mutations affecting induced mutagenesis in yeast

Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4–38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4⁺ and REV5⁺ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.     

拟南芥根毛功能基因AtGDPD-Like3关键氨基酸位点鉴定

AtGDPD-Like3是编码甘油磷酸二酯磷酸二酯酶（GDPD）类似基因,拟南芥该家族基因AtGDPD-Like3突变体shv3存在严重的根毛发育缺陷。为了鉴定AtGDPD-Like3关键氨基酸位点,我们构建S⁵³⁸A、V⁵⁵⁶A和D⁶²⁸A单点突变AtGDPD-Like3,分别转化atgdpdl3突变体并观察其恢复根毛缺陷程度。结果显示V⁵⁵⁶A、D⁶²⁸A位点突变AtGDPD-Like3完全恢复atgdpdl3根毛生长缺陷表型,但S⁵³⁸A突变AtGDPD-Like3只是部分恢复根毛缺陷。这些结果表明Ser⁵³⁸是AtGDPD-Like3较为关键的氨基酸位点,突变影响其蛋白质功能行使,同时暗示AtGDPD-Like3还存在其它的关键氨基酸位点。此研究结果为进一步探究AtGDPD-Like3蛋白功能行使的作用机制奠定了基础。     

MALE STERILITY 3 encodes a plant homeodomain-finger protein for male fertility in soybean

Male-sterile plants are used in hybrid breeding to improve yield in soybean (Glycine max (L.) Merr.). Developing the capability to alter fertility under different environmental conditions could broaden germplasm resources and simplify hybrid production. However, molecular mechanisms potentially underlying such a system in soybean were unclear. Here, using positional cloning, we identified a gene, MALE STERILITY 3 (MS3), which encodes a nuclear-localized protein containing a plant homeodomain (PHD)-finger domain. A spontaneous mutation in ms3 causing premature termination of MS3 translation and partial loss of the PHD-finger. Transgenetic analysis indicated that MS3 knockout resulted in nonfunctional pollen and no self-pollinated pods, and RNA-seq analysis revealed that MS3 affects the expression of genes associated with carbohydrate metabolism. Strikingly, the fertility of mutant ms3 can restore under long-dconditions. The mutant could thus be used to create a new, more stable photoperiod-sensitive genic male sterility line for two-line hybrid seed production, with significant impact on hybrid breeding and production.     

Involvement of recQ in the ultraviolet damage repair pathway in Deinococcus radiodurans

Deinococcus radiodurans is a bacterium which can survive extremely DNA damage. To investigate the relationship between recQ and the ultraviolet radiation (UV) damage repair pathway, we created a four mutant strain by constructing recQ knockout mutants in uvrA1, uvrA2, and uvsE backgrounds. Using the rpoB/Rif^r system, we measured the mutation frequencies and rates in wild type, recQ (MQ), uvsE uvrA1 uvrA2 (TNK006), and uvsE uvrA1 uvrA2 recQ (TQ). We then isolated Rif^r mutants of these strains and sequenced the rpoB gene. The mutation frequency of TQ was 6.4, 10.1, and 2.43 times that of wild type, MQ, and TNK006, respectively, and resulted in rates of 4.7, 6.71, and 2.15 folds higher than that of wild type, MQ, and TNK006, respectively. All the strains demonstrated specific mutational hotspots. Furthermore, the TQ strain showed a transversion bias that was different from the other three strains. The results indicate that recQ is involved in the ultraviolet damage repair pathway via the interaction between recQ and uvrA1, uvrA2, and uvsE in D. radiodurans.     

Arabidopsis Trithorax histone methyltransferases are redundant in regulating development and DNA methylation

Although the Trithorax histone methyltransferases ATX1–5 are known to regulate development and stress responses by catalyzing histone H3K4 methylation in Arabidopsis thaliana, it is unknown whether and how these histone methyltransferases affect DNA methylation. Here, we found that the redundant ATX1–5 proteins are not only required for plant development and viability but also for the regulation of DNA methylation. The expression and H3K4me3 levels of both RNA-directed DNA methylation (RdDM) genes (NRPE1, DCL3, IDN2, and IDP2) and active DNA demethylation genes (ROS1, DML2, and DML3) were downregulated in the atx1/2/4/5 mutant. Consistent with the facts that the active DNA demethylation pathway mediates DNA demethylationmainly at CG and CHG sites, and that the RdDM pathway mediates DNA methylation mainly at CHH sites, whole-genome DNA methylation analyses showed that hyper-CG and CHG DMRs in atx1/2/4/5 significantly overlapped with those in the DNA demethylation pathway mutant ros1 dml2 dml3 (rdd), and that hypo-CHH DMRs in atx1/2/4/5 significantly overlapped with those in the RdDM mutant nrpe1, suggesting that the ATX paralogues function redundantly to regulate DNA methylation by promoting H3K4me3 levels and expression levels of both RdDM genes and active DNA demethylation genes. Given that the ATX proteins function as catalytic subunits of COMPASS histone methyltransferase complexes, we also demonstrated that the COMPASS complex components function as a whole to regulate DNA methylation. This study reveals a previously uncharacterized mechanism underlying the regulation of DNA methylation.     

Influence of DNA repair on mutation spectra in Salmonella

This paper reviews the influence of DNA repair on spontaneous and mutagen-induced mutation spectra at the base-substitution (hisG46) and -1 frameshift (hisD3052) alleles present in strains of the Salmonella (Ames) mutagenicity assay. At the frameshift allele (mostly a CGCGCGCG target), ΔuvrB influences the frequency of spontaneous hotspot mutations (−CG), duplications, and deletions, and it also shifts the sites of deletions and duplications. Cells with pKM101+ΔuvrB spontaneously produce complex frameshifts (frameshifts with an adjacent base substitution). The spontaneous frequency of 1-base insertions or concerted (templated) mutations is unaffected by DNA repair, and neither mutation is inducible by mutagens. Glu-P-1, 1-nitropyrene (1NP), and 2-acetylaminofluorene (2AAF) induce only hotspot mutations and are unaffected by pKM101, whereas benzo(a)pyrene and 4-aminobiphenyl induce only hotspot in pKM101⁻, and hotspot plus complex in pKM101⁺. At the base-substitution allele (mostly a CC/GG target), the ΔuvrB allele increases spontaneous transitions in the absence of pKM101 and increases transversions in its presence. The frequency of suppressor mutations is decreased 4× by ΔuvrB, but increased 7.5× by pKM101. Both repair factors cause a shift in the proportion of mutations to the second position of the CC/GG target. With UV light and γ-rays, the ΔuvrB allele increases the proportion of transitions relative to transversions. pKM101 is required for mutagenesis by Glu-P-1 and 4-AB, and the types and positions of the substitutions are not altered by the addition of the ΔuvrB allele. Changes in DNA repair appear to cause more changes in spontaneous than in mutagen-induced mutation spectra at both alleles. There is a high correlation (r²=0.8) between a mutagen's ability to induce complex frameshifts and its relative base-substitution/frameshift mutagenic potency. A mutagen induces the same primary class of base substitution in TA100 (ΔuvrB, pKM101) as it does in Escherichia coli, mammalian cells, or rodents as well as in the p53 gene of human tumors associated with exposure to that mutagen. Thus, a mutagen induces the same primary class of base substitution in most organisms, reflecting the conserved nature of DNA replication and repair processes.     

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt^+/−→aprt^−/−) was 1.3 × 10⁻⁵/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt^−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.     

New mutations in cloned Escherichia coli umuDC genes: novel phenotypes of strains carrying a umuC125 plasmid

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD⁺C⁺ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD⁺C⁺ plasmids. pBR322-derived umuD⁺C⁺ plasmids inhibited the induction of the SOS response of lexA⁺ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA⁺ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA⁺ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD⁺C⁺ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.     

磷酸肌醇激酶FAB1调控拟南芥根毛伸长

FAB1/PIKfyve是介导PI(3,5)P₂ (磷脂酰肌醇3,5-二磷酸)生物合成的磷酸肌醇激酶。在动物和酵母(Saccharomyces cerevisiae)中, PI(3,5)P₂参与调控胞内膜运输, 但在植物中的研究较少。该文通过分析拟南芥(Arabidopsis thaliana) FAB1的T-DNA插入突变体的表型解析PI(3,5)P₂的生物学功能。拟南芥FAB1基因家族包含FAB1A、FAB1B、FAB1C和FAB1D四个基因。研究发现, fab1a/b呈现雄配子体致死的表型。利用遗传杂交获得fab1b/c/d三突变体, 发现FAB1B、FAB1C和FAB1D功能缺失导致根毛相比野生型变短, 经FAB1特异性抑制剂YM201636处理后的野生型中也观察到相似的短根毛表型。此外, fab1b/c/d三突变体中DR5转录水平降低。同时, 外源施加生长素类似物2,4-D和NAA能部分恢复fab1b/c/d植株短根毛的表型, 但fab1b/c/d突变体对生长素转运抑制剂(1-NOA和TIBA)的敏感性与野生型相似。此外, FAB1B/C/D功能缺失使根毛中ROS的含量减少且影响肌动蛋白的表达。上述结果表明, FAB1B/C/D通过调控生长素分布、ROS含量和肌动蛋白的表达影响拟南芥根毛伸长。     

一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

水稻(Oryza sativa)籽粒大小是影响其产量的关键农艺性状, 克隆并研究水稻籽粒大小相关基因对于提高水稻产量具有重要意义。为深入探究水稻籽粒大小的调控机制, 通过EMS诱变品种宽叶粳(KYJ), 分离了一系列水稻籽粒大小改变的突变体, 其中smg12表现为籽粒变小, 株高变矮, 一级枝梗数和二级枝梗数减少。遗传分析表明, 该小粒突变体受隐性单基因控制。细胞学分析显示, 该突变体颖壳纵向细胞长度显著变短, 表明SMG12主要影响细胞扩展。利用Mutmap方法对候选基因进行克隆, 筛选出SMG12的候选基因OsBRI1, 该基因编码油菜素内酯受体激酶。OsBRI1外显子上的第2 074个碱基发生了由C到T的置换, 产生非同义突变, 使得该位置编码的脯氨酸变为丝氨酸, 从而影响OsBRI1的功能。综上, 该研究鉴定了OsBRI1基因的1个新等位变异, 揭示了油菜素内酯途径调控水稻籽粒大小的细胞和分子基础。     

Threonine Overproduction in Yeast Strains Carrying the HOM3-R2 Mutant Allele under the Control of Different Inducible Promoters

The HOM3 gene of Saccharomyces cerevisiae codes for aspartate kinase, which plays a crucial role in the regulation of the metabolic flux that leads to threonine biosynthesis. With the aim of obtaining yeast strains able to overproduce threonine in a controlled way, we have placed the HOM3-R2 mutant allele, which causes expression of a feedback-insensitive enzyme, under the control of four distinctive regulatable yeast promoters, namely, P_GAL1, P_CHA1, P_CYC1-HSE2, and P_GPH1. The amino acid contents of strains bearing the different constructs were analyzed both under repression and induction conditions. Although some differences in overall threonine production were found, a maximum of around 400 nmol/mg (dry weight) was observed. Other factors, such as excretion to the medium and activity of the catabolic threonine/serine deaminase, also affect threonine accumulation. Thus, improvement of threonine productivity by yeast cells would probably require manipulation of these and other factors.     

Mutation or deletion of the Saccharomyces cerevisiae RAT3/NUP133 gene causes temperature-dependent nuclear accumulation of poly(A)+ RNA and constitutive clustering of nuclear pore complexes.

To identify genes whose products play potential roles in the nucleocytoplasmic export of messenger RNA, we isolated temperature-sensitive strains of Saccharomyces cerevisiae and examined them by fluorescent in situ hybridization. With the use of a digoxigen-tagged oligo-(dT)50 probe, we identified those that showed nuclear accumulation of poly(A)+ RNA when cells were shifted to the nonpermissive temperature. We describe here the properties of yeast strains bearing the rat3-1 mutation (RAT-ribonucleic acid trafficking) and the cloning of the RAT3 gene. When cultured at the permissive temperature of 23 degrees C, fewer than 10% of cells carrying the rat3-1 allele showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA, whereas approximately 70% showed nuclear accumulation of poly(A)+ RNA after a shift to 37 degrees C for 4 h. In wild-type cells, nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope. Both indirect immunofluorescence analysis and electron microscopy of rat3-1 cells indicated that NPCs were clustered into one or a few regions of the NE in mutant cells. Similar NPC clustering was seen in mutant cells cultured at temperatures between 15 degrees C and 37 degrees C. The RAT3 gene encodes an 1157-amino acid protein without similarity to other known proteins. It is essential for growth only at 37 degrees C. Cells carrying a disruption of the RAT3 gene were very similar to cells carrying the original rat3-1 mutation; they showed temperature-dependent nuclear accumulation of poly(A)+ RNA and exhibited constitutive clustering of NPCs. Epitope tagging of Rat3p demonstrated that it is located at the nuclear periphery and co-localizes with nuclear pore proteins recognized by the RL1 monoclonal antibody. We refer to this nucleoporin as Rat3p/Nup133p.     

人β-半乳糖苷酶R299L突变体的获得与活性研究*

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。     

Utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora for risk assessment of environmental chemicals.

The utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora crassa is compared with that of other eukaryotic assay systems for the evaluation of the mutagenic effects of environmental chemicals. In contrast to other in vitro specific-locus assays, the Neurospora assay can detect mutations not only at the ad-3A and ad-3B loci but also recessive lethal mutations elsewhere in the genome. Mutational damage in this system can be characterized readily by means of classical genetic techniques involving heterokaryon tests to determine genotype, and allelic complementation among ad-3B^R mutations. The percentages of ad-3B^R mutations showing allelic complementation with polarized or nonpolirized complementation patterns provide a presumptive identification of the genetic alterations at the molecular level in individual mutants. Dikaryon and trikaryon tests (using 3 strains carrying multilocus deletion mutations as tester strains) distinguish ad-3 mutations resulting from gene/point mutation, multilocus deletion mutation, and various types of multiple-locus mutation.

The array of ad-3 mutations recovered from forward-mutation experiments can be expressed in terms of Mutational Spectra, which make it possible to make comparisons of mutational types between different doses of the same mutagen, different mutagens, or the effects of the same mutagen on different strains.

Another important feature of this specific-locus assay system is that the effects of mutagens can be studied in both DNA excision repair-proficient (H-12) and -deficient (H-59) two-component heterokaryons to evaluate both quantitative and qualitative differences between the spectra of induced d-3     

Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, ΔuvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage—resulting in an antimutagenic effect on spontaneous mutation.