Possible regulatory function of the Saccharomyces cerevisiae Ty1 retrotransposon core protein

The yeast Ty1 retrotransposon encodes proteins and RNA that assemble into virus-like particles (VLPs) as part of the life cycle of the retro-element. The Tya protein, which is equivalent to the retroviral Gag, is the major structural component of these particles. In this work, we demonstrate that Tya proteins fulfil other functions apart from their structural role. We show that Tya interacts in vitro with the Ty1 RNA domain required for RNA packaging, suggesting that this RNA-protein interaction may direct the packaging process. Furthermore, the overexpression of both Tya proteins, i.e. p1, the primary translation product, and p2, the mature form, increases endogenous Ty1 RNA levels in trans without increasing translation significantly. These observations suggest that Tya may exert a regulatory function during transposition. Interestingly, however, only p2, the mature form of Tya, trans-activates transposition of a marked genomic Ty element. This confirms that processing is required for transposition.     

The yeast Ty virus-like particles

Virus-like particle (VLP) assembly is a crucial step of the life cycle of retrotransposons. The S. cerevisiae Ty elements represent an interesting model for the analysis of these particles and thus have been studied extensively. Our current knowledge of the organisation and assembly of Ty1 and Ty3 VLPs is reviewed here. This includes the mechanism of assembly, the role of the Tya core protein during VLP formation and the RNA packaging process. The physical properties of Ty1 VLPs are also described and the latest three-dimensional Ty1 VLP reconstructions are shown. In addition, the relevance of these studies is discussed in the context of retro-element biology.     

Analysis of a Ty1-less variant of Saccharomyces paradoxus: the gain and loss of Ty1 elements

Because Ty elements transpose through an RNA intermediate, element accumulation through retrotransposition must be regulated or offset by element loss to avoid uncontrolled genome expansion. Here we examine the fate of Ty sequences in Saccharomyces strain 337, a strain that is reported to lack Ty1 and Ty2 elements, but contains remnant solo long terminal repeats (LTRs). Although strain 337 was initially classified as Saccharomyces cerevisiae, our work indicates that this strain is more closely related to S. paradoxus. Several degenerate Ty1 and Ty2 LTRs were mapped to the same insertion sites as full-length Ty1 and Ty2 elements in S. cerevisiae, suggesting that this strain lost Ty elements by LTR-LTR recombination. Southern analysis indicates that strain 337 also lacks Ty4 and Ty5 elements. We estimated the rates of element gain and loss in this strain by introducing a single transposition-competent Ty1 element. The results indicate that Ty1 retrotransposition occurs at a much higher rate than elimination, suggesting that copy-number-dependent co-factors or environmental conditions contribute to the loss of Ty elements in this genome.     

The role of reactive oxygen species in the induction of Ty1 retrotransposition in Saccharomyces cerevisiae

Here we provide evidence for a dependence between the increased production of reactive oxygen species and the activation of Ty1 retrotransposition. We have found that the strong activator of Ty1 mobility, methylmethane sulphonate, can not induce Ty1 retrotransposition in cells with compromised mitochondrial oxidative phosphorylation (rho^?; sco1Δ), which is the major source for production of reactive oxygen species (ROS) in Saccharomyces cerevisiae. The quantitative estimation of superoxide anions in living cells showed that rho⁺ cells exposed to methylmethane sulphonate increase Ty1 retrotransposition and superoxide levels. The increase of superoxide anions by the superoxide generator menadione is accompanied by induction of Ty1 mobility without any treatment with a DNA‐damaging agent. Higher frequencies of retrotransposition were found in rho⁺ and rho^? cells treated with exogenously added hydrogen peroxide or in cells with disrupted YAP1 gene characterized by increased intracellular levels of hydrogen peroxide. These data indicate that increased levels of ROS may have an independent and key role in the induction of Ty1 retrotransposition. Copyright © 2010 John Wiley & Sons, Ltd.     

多酚类化合物基于非编码RNA调控发挥抗肿瘤及辐射增敏作用研究进展

恶性肿瘤严重危害人类生命健康，放疗是目前治疗恶性肿瘤常用的方法之一。然而电离辐射会引起肿瘤细胞的辐射抗性并损伤正常组织细胞，限制了其在临床上的应用。多酚类天然产物不仅可以提高肿瘤细胞的辐射敏感性，同时又能缓解辐射对机体产生的负面效应，在肿瘤治疗中发挥着重要作用。非编码RNA在许多疾病的发生发展中起着重要作用，近年来研究发现多酚类物质在抗肿瘤及辐射增敏方面与非编码RNA的调控存在一定关联。基于此，本文综述天然产物多酚抗肿瘤和辐射增敏的作用及其机制，并基于非编码RNA调控层面阐释其作用效果，以期为肿瘤等疾病的干预治疗及相关特医膳食食品的开发提供新思路。     

Distribution and sequence analysis of a novel Ty3-like element in natural Saccharomyces paradoxus isolates

Little is known about the transposable elements of species closely related to Saccharomyces cerevisiae. We present a novel transposable element in Saccharomyces paradoxus, a close congener of S. cerevisiae. Sequence analysis of this element, designated Ty3-1p, indicates that it is a homologue of the S. cerevisiae Ty3 element. Ty3-1p shares 82% nucleotide identity with an S. cerevisiae Ty3 element and appears to be structured identically to Ty3, containing two overlapping open reading frames, six retroviral-like domains, a J domain, and flanking sigma-like elements. A sigma element from Ty3-1p is 75% identical to a Ty3 sigma element. There is no evidence of horizontal transfer of Ty3 in Saccharomyces sensu stricto. We assess the distributions of Ty3p and Ty3 element insertions in natural population samples of S. paradoxus and S. cerevisiae. The S. paradoxus population sample exhibits Ty3p insertions present at a variety of sites at low frequency; this suggests that Ty3p elements are active in the sampled population. The S. cerevisiae population sample exhibits a uniform Ty3 hybridization profile in which all element insertions appear to be fixed. We comment on the possible causes of these contrasting observed distributions (GenBank Accession Nos AY198186 and AY198187).     

Improvement and induction property of radiation-responsive promoter through DNA shuffling of 5′-flanking regions of the human p21 gene

A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy γ-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-κB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.     

Effect of citric acid on the radiation resistance of Listeria monocytogenes and frankfurter quality factors

Listeria monocytogenes is a common contaminant of ready-to-eat meat products, including frankfurters. Ionizing (gamma) radiation can eliminate L. monocytogenes from frankfurters. Citric acid (CA) is an antioxidant synergist and anti-microbial agent that can be applied to the surfaces of cured meat products prior to packaging. The effect of CA on the radiation resistance of L. monocytogenes that was surface-inoculated onto frankfurters was determined. The D(10) values, the radiation doses required to inactivate 90% of viable L. monocytogenes, were 0.61, 0.60, 0.54, and 0.53 kGy, on frankfurters dipped in 0, 1, 5 or 10% CA solution, respectively. CA, although an antioxidant synergist, did not increase antioxidant activity (AA) on frankfurter surfaces as determined by the ferric reducing antioxidant power (FRAP) assay. Lipid oxidation, as determined by the Thiobarbituric acid reactive substances (TBARS) assay, was not affected by CA or ionizing radiation. Color of frankfurters, determined by Hunter L, a, b, indicated that ionizing radiation induced a small, but visually imperceptible, loss of redness (a-value). Frankfurter firmness, as measured by maximum shear force, was not affected by ionizing radiation or CA. CA enhanced the lethality of ionizing radiation without negatively impacting frankfurter color, lipid oxidation, firmness, or antioxidant activity.     

Inferences of evolutionary relationships from a population survey of LTR-retrotransposons and telomeric-associated sequences in the Saccharomyces sensu stricto complex

The Saccharomyces sensu stricto complex consists of six closely related species and one natural hybrid. Intra- and inter- species variability in repetitive elements can help elucidate the population structure and evolution of these close relatives. The chromosome positions of several telomeric associated sequences (TASs) and LTR-retrotransposons have been determined, using PFGE, in 112 isolates. Most of the repetitive elements studied are found in multiple copies in each strain, although in some subpopulations these elements are present in low copy number or are absent. Hybridization patterns and copy numbers of the repetitive elements correlate with geographic distribution. These patterns may yield interesting clues as to the origins and evolution of some TASs and retrotransposons, e.g. we can infer that Y' originated on the left end of chromosome XIV. There is strong evidence for horizontal transfer of Ty2 between S. cerevisiae and S. mikatae. Ty1 and Ty5 are either lost easily or frequently horizontally transferred. We have also found some gross chromosomal rearrangements in isolates within species and a few new natural hybrids between species, indicating that these processes occur in the wild and are not limited to conditions of human influence. DNA sequences have been deposited with the EMBL/GenBank database under Accession Nos AJ632279-AJ632293.     

Ty3/gypsy-like retrotransposons in Candida albicans and Candida dubliniensis: Tca3 and Tcd3

Ty3/gypsy retrotransposons are a widespread group of eukaryote mobile genetic elements. They are similar in structure to, and may be ancestors of, the vertebrate retroviruses. Here we describe the first Ty3/gypsy retrotransposons from the pathogenic yeasts Candida albicans and Candida dubliniensis, which we refer to as Tca3 and Tcd3, respectively. Tca3 was first identified in a variety of strains as an element lacking a large part of its coding region. Comparative analyses between C. albicans and C. dubliniensis allowed us to identify the closely related full-length Tcd3 element, and, subsequently, the full-length Tca3 elements. The full-length versions of Tca3 and Tcd3 are broadly similar in structure to other Ty3/gypsy elements, but have several features of special interest, e.g. both elements appear to have a novel mechanism for priming minus-strand DNA synthesis, probably involving conserved secondary structures adjacent to the 5' LTRs. Also, while closely related to each other, the two elements appear to be fairly distantly related to other known Ty3/gypsy-like elements. Finally, the occurrence of the internally deleted forms of Tca3 in many strains raises interesting questions concerning the evolution of these transposable elements in Candida and the evolution of Candida itself. The sequences reported in this paper have been assigned GenBank Accession Nos AF499463, AF499464 and AF510498.     

提取猕猴桃果实总RNA的新方法

目的:获得1种提取猕猴桃果实总RNA的简单、快速、高效的方法。方法:使用异硫氰酸胍提取剂,同时在提取剂中加入1/10体积的5mol/L醋酸钾及1/4体积的100%冰乙醇提取猕猴桃果实总RNA。将经检测合格的RNA反转录为cDNA。以上述cDNA为模板,通过PCR方法扩增猕猴桃果胶甲基酯酶抑制剂基因。结果:所提取的猕猴桃果实总RNA可完全消除果实组织多糖的干扰,并在实验室成功地用于cDNA的合成和PCR扩增反应等。结论:与其它提取方法相比,本方法具有操作步骤简单、耗时少等优点,还可用于其它富含多糖的果实组织(如番茄等)的RNA提取。     

圆弧青霉脂肪酶基因的克隆和表达

圆弧青霉PG37总RNA甲醛变性电泳呈现真核生物所特有的 2 8SrRNA和 18SrRNA条带 .根据PG37所产碱性脂肪酶 (LipPC)N末端氨基酸残基序列和真核生物mRNA在 3’端具有poly(A)等所提供的生物信息 ,采用RT PCR技术扩增了LipPC成熟肽和 3’非编码区的cDNA片段 ,并将该PCR产物直接克隆至pUCm T载体中 .序列分析表明 ,LipPC含有 2 5 8个氨基酸残基 ,其中保守的五肽序列为Gly His Ser Leu Gly .进一步采用ClonTech公司的SmartTM PCRcDNA文库构建试剂盒 ,扩增、克隆和测定了自转录起始点至编码区的cDNA片段 ,从而完成了LipPC完整cDNA的分析测定 .最后将编码完整脂肪酶蛋白质的cDNA克隆至 pGEX 5X 3表达载体中 ,SDS PAGE检测结果表明 ,大肠杆菌BL2 1所表达的GST LipPC融合蛋白质分子质量约为 5 3ku .     

Impact of Ionizing Radiation and Thermal Treatments on Furan Levels in Fruit Juice

The formation of furan in freshly prepared apple and orange juices as affected by ionizing radiation and thermal treatments was studied using a newly developed solid‐phase microextraction method coupled with gas chromatography‐mass spectrometry (GC‐MS). Results show that furan levels increased linearly as radiation dose increased from 0 to 5 kGy. Irradiation induced more furan in apple juice than in orange juice. During post‐irradiation storage at 4 °C, furan levels increased in both apple and orange juices, particularly in the 1st 3 d. On the other hand, irradiation degraded deuterated furan (d₄‐furan) spiked in water and fruit juices. The rate of degradation as a function of radiation dose was the highest in water and the lowest in orange juice. Submerging the juice samples in boiling water for 5 min induced higher amounts of furan in orange juice than in apple juice, but autoclaving (121 °C, 25 min) resulted in more furan formation in apple juice than in orange juice. Results reported here suggest that both ionizing radiation and thermal treatments induce furan formation in fruit juices.