共查询到20条相似文献,搜索用时 31 毫秒
1.
Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion 总被引:8,自引:6,他引:2
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Zhang Y Wu X Rechkoblit O Geacintov NE Taylor JS Wang Z 《Nucleic acids research》2002,30(7):1630-1638
REV1 functions in the DNA polymerase ζ mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3′→5′ proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19–27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N 2-dG, (–)-trans-anti-benzo[a]pyrene-N 2-dG and 1,N 6-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6–4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase κ, bypass of the trans-anti-benzo[a]pyrene-N 2 -dG adducts and the 1,N 6-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass. 相似文献
2.
3.
In vitro and in vivo modulations of benzo[c]phenanthrene-DNA adducts by DNA mismatch repair system
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Wu J Zhu BB Yu J Zhu H Qiu L Kindy MS Gu L Seidel A Li GM 《Nucleic acids research》2003,31(22):6428-6434
Benzo[c]phenanthrene dihydrodiol epoxide (B[c] PhDE) is well known as an important environmental chemical carcinogen that preferentially modifies DNA in adenine residues. However, the molecular mechanism by which B[c]PhDE induces tumorigenesis is not fully understood. In this report, we demonstrate that DNA mismatch repair (MMR), a genome maintenance system, plays an important role in B[c]PhDE-induced carcinogensis by promoting apoptosis in cells treated with B[c]PhDE. We show that purified human MMR recognition proteins, MutSα and MutSβ, specifically recognized B[c]PhDE-DNA adducts. Cell lines proficient in MMR exhibited several-fold more sensitivity to killing than cell lines defective in either MutSα or MutLα by B[c]PhDE; the nature of this sensitivity was shown to be due to increased apoptosis. Additionally, wild-type mice exposed to B[c]PhDE had intestinal crypt cells that underwent apoptosis significantly more often than intestinal crypt cells found in B[c]PhDE-treated Msh2–/– or Mlh1–/– mice. These findings, combined with previous studies, suggest that the MMR system may serve as a general sensor for chemical-caused DNA damage to prevent damaged cells from mutagenesis and carcinogenesis by promoting apoptosis. 相似文献
4.
cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from Pseudomonas sp. Strain P51, Expressed in Escherichia coli DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Henning Raschke Thomas Fleischmann Jan Roelof Van Der Meer Hans-Peter E. Kohler 《Applied microbiology》1999,65(12):5242-5246
cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5α(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (−)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (−)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols. 相似文献
5.
Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Frank EG Sayer JM Kroth H Ohashi E Ohmori H Jerina DM Woodgate R 《Nucleic acids research》2002,30(23):5284-5292
Human DNA polymerase ι (polι) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of polι to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to polι-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 × 10–3 and 6 × 10–4. Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polκ, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that polι might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polκ. 相似文献
6.
Hexamminecobalt(III)-induced
condensation of calf thymus DNA: circular dichroism and hydration measurements 总被引:2,自引:2,他引:0
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The interaction of hexamminecobalt(III), Co(NH3)63+, with 160 and 3000–8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH3)63+ binding up to the molar ratio [Co(NH3)63+]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH3)63+]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH3)63+]/[P] = 0.3. In the case of 3000–8000 bp DNA only two processes were observed: (i) binding up to [Co(NH3)63+]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B→Ψ transition after [Co(NH3)63+]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH3)63+ binding on volume and compressibility have been obtained. The binding of Co(NH3)63+ to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH3)63+: ΔV = 9 cm3 mol–1 and Δκ = 33 × 10–4 cm3 mol–1 bar–1. The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH3)63+ and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA. 相似文献
7.
Mitochondria are major cellular targets of benzo[a]pyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N2-deoxyguanosine (dG) and N6-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzo[c]phenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol γ) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DE–dG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DE–dA adducts derived from both BaP and BcPh. In the presence of p55 the polymerase incorporated all four nucleotides and catalyzed limited translesion synthesis past BaP DE–dG adducts but not past BaP or BcPh DE–dA adducts. Thus, all these adducts cause erroneous purine incorporation and significant blockage of further primer elongation. Purine misincorporation by pol γ opposite the BaP DE–dG adducts resembles that observed with the Y family pol η. Blockage of translesion synthesis by these DE adducts is consistent with known BaP inhibition of mitochondrial (mt)DNA synthesis and suggests that continued exposure to BaP reduces mtDNA copy number, increasing the opportunity for repopulation with pre-existing mutant mtDNA and a resultant risk of mitochondrial genetic diseases. 相似文献
8.
Sabine Lutz-Wahl Peter Fischer Claudia Schmidt-Dannert Wolfgang Wohlleben Bernhard Hauer Rolf D. Schmid 《Applied microbiology》1998,64(10):3878-3881
A total of 215 Streptomyces strains were screened for their capacity to regio- and stereoselectively hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives. With β-ionone as the substrate, 15 strains showed little conversion to 4-hydroxy- and none showed conversion to the 3-hydroxy product as desired. Among these 15 Streptomyces strains, S. fradiae Tü 27, S. arenae Tü 495, S. griseus ATCC 13273, S. violaceoniger Tü 38, and S. antibioticus Tü 4 and Tü 46 converted α-ionone to 3-hydroxy-α-ionone with significantly higher hydroxylation activity compared to that of β-ionone. Hydroxylation of racemic α-ionone [(6R)-(−)/(6S)-(+)] resulted in the exclusive formation of only the two enantiomers (3R,6R)- and (3S,6S)-hydroxy-α-ionone. Thus, the enzymatic hydroxylation of α-ionone by the Streptomyces strains tested proceeds with both high regio- and stereoselectivity. 相似文献
9.
Zhang Y Yuan F Wu X Rechkoblit O Taylor JS Geacintov NE Wang Z 《Nucleic acids research》2000,28(23):4717-4724
DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase η (Polη), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Polη, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Polη efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Polη effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5′ to the abasic site is a T. Human Polη partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N2-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N2-dG lesion in mammalian cells. These results show that human Polη is capable of error-prone translesion DNA syntheses in vitro and suggest that Polη may bypass certain lesions with a mutagenic consequence in humans. 相似文献
10.
12-Oxophytodienoate-10,11-Reductase: Occurrence of Two Isoenzymes
of Different Specificity against Stereoisomers of 12-Oxophytodienoic
Acid 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The reduction of 12-oxophytodienoic acid (OPDA) to 3-oxo-2(2′[Z]-pentenyl)-cyclopentane-1-octanoic acid is catalyzed by 12-oxophytodienoate-10,11-reductase (OPR). Analysis of the isomer preference of OPR has indicated that the activity is composed of two isoenzymes exhibiting different stereoselectivities. The two isoforms of OPR have been separated, using protein extracts of Rock Harlequin (Corydalis sempervirens) as the starting material. OPRI, the enzyme reported earlier from the same species and corresponding to the cloned OPR from Arabidopsis, utilized 9R,13R-OPDA >> 9S,13R-OPDA but not the 13S-configured isomers, whereas the new activity, OPRII, effectively reduced all four OPDA isomers, including the natural 9S,13S-OPDA (cis-[+]-OPDA). OPRII activity is characterized in detail. The enzyme's enzymatic, biochemical, and immunological properties prove that it is a close relative of OPRI. The roles of OPRI and OPRII in octadecanoid biology are discussed. 相似文献
11.
DNA bypass polymerases are utilized to transit bulky DNA lesions during replication, but the process frequently causes mutations. The structural origins of mutagenic versus high fidelity replication in lesion bypass is therefore of fundamental interest. As model systems, we investigated the molecular basis of the experimentally observed essentially faithful bypass of the guanine 10S-(+)-trans-anti-benzo[a]pyrene-N2-dG adduct by the Y-family human DNA polymerase κ, and the observed blockage of pol κ produced by the adenine 10S-(+)-trans-anti-benzo[a]pyrene-N2-dA adduct. These lesions are derived from the most tumorigenic metabolite of the ubiquitous cancer-causing pollutant, benzo[a]pyrene. We compare our results for the dG adduct with our earlier studies for the pol κ archaeal homolog Dpo4, which processes the same lesion in an error-prone manner. Molecular modeling, molecular mechanics calculations and molecular dynamics simulations were utilized. Our results show that the pol κ N-clasp is a key structural feature that accounts for the dA adduct blockage and the near-error-free bypass of the dG lesion. Absence of the N-clasp in Dpo4 explains the error-prone processing of the same lesion by this enzyme. Thus, our studies elucidate structure-function relationships in the fidelity of lesion bypass. 相似文献
12.
Benzo[a]pyrene is an important environmental mutagen and carcinogen. Its metabolism in cells yields the mutagenic, key ultimate carcinogen 7R,8S,9S,10R-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, (+)-anti-BPDE, which reacts via its 10-position with N2-dG in DNA to form the adduct (+)-trans-anti-BPDE-N2-dG. To gain molecular insights into BPDE-induced mutagenesis, we examined in vivo translesion synthesis and mutagenesis in yeast cells of a site-specific 10S (+)-trans-anti-BPDE-N2-dG adduct and the stereoisomeric 10R (−)-trans-anti-BPDE-N2-dG adduct. In wild-type cells, bypass products consisted of 76% C, 14% A and 7% G insertions opposite (+)-trans-anti-BPDE-N2-dG; and 89% C, 4% A and 4% G insertions opposite (−)-trans-anti-BPDE-N2-dG. Translesion synthesis was reduced by ~26–37% in rad30 mutant cells lacking Polη, but more deficient in rev1 and almost totally deficient in rev3 (lacking Polζ) mutants. C insertion opposite the lesion was reduced by ~24–33% in rad30 mutant cells, further reduced in rev1 mutant, and mostly disappeared in the rev3 mutant strain. The insertion of A was largely abolished in cells lacking either Polη, Polζ or Rev1. The insertion of G was not detected in either rev1 or rev3 mutant cells. The rad30 rev3 double mutant exhibited a similar phenotype as the single rev3 mutant with respect to translesion synthesis and mutagenesis. These results show that while the Polζ pathway is generally required for translesion synthesis and mutagenesis of the (+)- and (−)-trans-anti-BPDE-N2-dG DNA adducts, Polη, Polζ and Rev1 together are required for G→T transversion mutations, a major type of mutagenesis induced by these lesions. Based on biochemical and genetic results, we present mechanistic models of translesion synthesis of these two DNA adducts, involving both the one-polymerase one-step and two-polymerase two-step models. 相似文献
13.
Enantioselective Uptake and Degradation of the Chiral Herbicide Dichlorprop [(RS)-2-(2,4-Dichlorophenoxy)propanoic acid] by Sphingomonas herbicidovorans MH
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Christian Zipper Monika Bunk Alexander J. B. Zehnder Hans-Peter E. Kohler 《Journal of bacteriology》1998,180(13):3368-3374
Sphingomonas herbicidovorans MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop [(RS)-2-(2,4-dichlorophenoxy)propanoic acid], with preferential degradation of the (S) enantiomer over the (R) enantiomer. These results are in agreement with the recently reported enantioselective degradation of mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid] by this bacterium (C. Zipper, K. Nickel, W. Angst, and H.-P. E. Kohler, Appl. Environ. Microbiol. 62:4318–4322, 1996). Uptake of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible. Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (Kt) of 108, 93, and 117 μM and maximal velocities (Vmax) of 19, 10, and 21 nmol min−1 mg of protein−1 for (R)-dichlorprop, (S)-dichlorprop, and 2,4-D, respectively. Transport of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor N,N′-dicyclohexylcarbodiimine. Experiments on the substrate specificity of the putative transport systems revealed that (R)-dichlorprop uptake was inhibited by (R)-mecoprop but not by (S)-mecoprop, (S)-dichlorprop, or 2,4-D. On the other hand, the (S)-dichlorprop transport was inhibited by (S)-mecoprop but not by (R)-mecoprop, (R)-dichlorprop, or 2,4-D. These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by S. herbicidovorans is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (R)-dichlorprop and (R)-mecoprop, another for (S)-dichlorprop and (S)-mecoprop, and a third for 2,4-D. 相似文献
14.
Hydration effects of Ni(2+) binding to synthetic polynucleotides with regularly alternating purine-pyrimidine sequences
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Kankia BI 《Nucleic acids research》2000,28(4):911-916
High precision ultrasonic and densimetric techniques have been used to study the interaction of Ni2+ ions with right-handed poly[d(G-C)]·poly[d(G-C)], poly-[d(A-C)]·poly[d(G-T)] and poly[d(A-T)]·poly[d(A-T)] in 5 mM CsCl, 0.2 mM HEPES, pH 7.5 at 20°C. From these measurements the changes in the apparent molar volume and the apparent molar adiabatic compressibility due to the interaction have been obtained. The volume effects of the binding, calculated per mole of Ni2+ ions, range from 11.7 to 23.9 cm3 mol–1 and the compressibility effects range from 19.3 × 10–4 to 43.1 × 10–4 cm3 mol–1 bar–1. These data are interpreted in terms of dehydration of the polynucleotides and Ni2+ ions, i.e. the release of water molecules from the hydration shells of the molecules. An increase in G+C content gives an increase in volume and compressibility effects, indicating a rise in the extent of dehydration. The dehydration effects of Ni2+ binding to poly[d(G-C)]·poly[d(G-C)] are approximately twice those of poly[d(A-T)]·poly[d(A-T)]. The volume and compressibility effects of Ni2+–EDTA complex formation have also been measured and used as a model system for quantitative estimation. These values revealed that Ni2+ ions can coordinate two atomic groups of poly[d(G-C)]·poly[d(G-C)], while in the case of the Ni2+–poly[d(A-T)]·poly[d(A-T)] complex volume and compressibility effects correspond to one direct or two indirect (through water) contacts. 相似文献
15.
A crystallographic study of the binding of 13 metal ions to two related RNA duplexes 总被引:5,自引:3,他引:2
Metal ions, and magnesium in particular, are known to be involved in RNA folding by stabilizing secondary and tertiary structures, and, as cofactors, in RNA enzymatic activity. We have conducted a systematic crystallographic analysis of cation binding to the duplex form of the HIV-1 RNA dimerization initiation site for the subtype-A and -B natural sequences. Eleven ions (K+, Pb2+, Mn2+, Ba2+, Ca2+, Cd2+, Sr2+, Zn2+, Co2+, Au3+ and Pt4+) and two hexammines [Co (NH3)6]3+ and [Ru (NH3)6]3+ were found to bind to the DIS duplex structure. Although the two sequences are very similar, strong differences were found in their cation binding properties. Divalent cations bind almost exclusively, as Mg2+, at ‘Hoogsteen’ sites of guanine residues, with a cation-dependent affinity for each site. Notably, a given cation can have very different affinities for a priori equivalent sites within the same molecule. Surprisingly, none of the two hexammines used were able to efficiently replace hexahydrated magnesium. Instead, [Co (NH3)4]3+ was seen bound by inner-sphere coordination to the RNA. This raises some questions about the practical use of [Co (NH3)6]3+ as a [Mg (H2O)6]2+ mimetic. Also very unexpected was the binding of the small Au3+ cation exactly between the Watson–Crick sites of a G-C base pair after an obligatory deprotonation of N1 of the guanine base. This extensive study of metal ion binding using X-ray crystallography significantly enriches our knowledge on the binding of middleweight or heavy metal ions to RNA, particularly compared with magnesium. 相似文献
16.
1. Rhodesian copalwood (Guibourtia coleosperma) contains three diastereo-isomeric leuco-fisetinidins. These consist of the (−)-2,3-cis–3,4-cis (2R,3R,4R) and (−)-2,3-cis–3,4-trans (2R,3R,4S) 3′,4′,7-trihydroxyflavan-3,4-diols, and the third was shown to be a 2,3-trans–3,4-cis isomer by means of paper ionophoresis. 2. There occurrence in similar proportions as tannin precursors also in the tropical hardwoods G. tessmannii and G. demeusii implies a close taxonomic relationship between these, and with G. coleosperma. 3. Epimerization of the natural (−)-3′,4′,7- trihydroxy-2,3-trans-flavan-3,4-trans-diol affords a mixture from which the (−)-2,3-cis–3,4-cis isomer was separated readily, but the (−)-2,3-trans–3,4-cis isomer was obtained with difficulty. These were formed by epimerization of the (−)-2,3-trans–3,4-trans isomer at C-2 and C-4, and at C-4, respectively. 相似文献
17.
Syntheses and structural studies of calix[4]arene-nucleoside and calix[4]arene-oligonucleotide hybrids
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have synthesized three types of calix[4]arene– nucleoside hybrid efficiently by amide bond formation between the amine functional groups of 1,3-diaminocalix[4]arene and the carboxyl groups of thymidine nucleoside derivatives. X-ray crystallography of a homocoupled calix[4]arene–nucleoside hybrid revealed an interesting hydrogen bonding pattern between thymine bases and the amide linkages. We designed the calix[4]arene–oligonucleotide hybrids (5′-AAAAGATATCAAXTTGATATCTTTT-3′, 5′-T12-X-T12-3′, and 5′-A12-X-T12-3′) to be V-shaped oligodeoxyribonucleotides and synthesized them by using a calix[4]arene–nucleoside hybrid (X) as a key building block. Thermal denaturation experiments, monitored by UV spectroscopy at 260 and 284 nm, and circular dichroism spectra of the calix[4]arene–oligonucleotide hybrids suggest that the modified oligonucleotides indeed adopt V-shaped conformations, making them suitable for use as building blocks in the construction of programmed oligonucleotide nanostructures. 相似文献
18.
Effect of Selected Monoterpenes on Methane Oxidation, Denitrification, and Aerobic Metabolism by Bacteria in Pure Culture 总被引:6,自引:2,他引:4
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Selected monoterpenes inhibited methane oxidation by methanotrophs (Methylosinus trichosporium OB3b, Methylobacter luteus), denitrification by environmental isolates, and aerobic metabolism by several heterotrophic pure cultures. Inhibition occurred to various extents and was transient. Complete inhibition of methane oxidation by Methylosinus trichosporium OB3b with 1.1 mM (−)-α-pinene lasted for more than 2 days with a culture of optical density of 0.05 before activity resumed. Inhibition was greater under conditions under which particulate methane monooxygenase was expressed. No apparent consumption or conversion of monoterpenes by methanotrophs was detected by gas chromatography, and the reason that transient inhibition occurs is not clear. Aerobic metabolism by several heterotrophs was much less sensitive than methanotrophy was; Escherichia coli (optical density, 0.01), for example, was not affected by up to 7.3 mM (−)-α-pinene. The degree of inhibition was monoterpene and species dependent. Denitrification by isolates from a polluted sediment was not inhibited by 3.7 mM (−)-α-pinene, γ-terpinene, or β-myrcene, whereas 50 to 100% inhibition was observed for isolates from a temperate swamp soil. The inhibitory effect of monoterpenes on methane oxidation was greatest with unsaturated, cyclic hydrocarbon forms [e.g., (−)-α-pinene, (S)-(−)-limonene, (R)-(+)-limonene, and γ-terpinene]. Lower levels of inhibition occurred with oxide and alcohol derivatives [(R)-(+)-limonene oxide, α-pinene oxide, linalool, α-terpineol] and a noncyclic hydrocarbon (β-myrcene). Isomers of pinene inhibited activity to different extents. Given their natural sources, monoterpenes may be significant factors affecting bacterial activities in nature. 相似文献
19.
Epigenetically inherited aggregates of the yeast prion [PSI+] cause genomewide readthrough translation that sometimes increases evolvability in certain harsh environments. The effects of natural selection on modifiers of [PSI+] appearance have been the subject of much debate. It seems likely that [PSI+] would be at least mildly deleterious in most environments, but this may be counteracted by its evolvability properties on rare occasions. Indirect selection on modifiers of [PSI+] is predicted to depend primarily on the spontaneous [PSI+] appearance rate, but this critical parameter has not previously been adequately measured. Here we measure this epimutation rate accurately and precisely as 5.8 × 10−7 per generation, using a fluctuation test. We also determine that genetic “mimics” of [PSI+] account for up to 80% of all phenotypes involving general nonsense suppression. Using previously developed mathematical models, we can now infer that even in the absence of opportunities for adaptation, modifiers of [PSI+] are only weakly deleterious relative to genetic drift. If we assume that the spontaneous [PSI+] appearance rate is at its evolutionary optimum, then opportunities for adaptation are inferred to be rare, such that the [PSI+] system is favored only very weakly overall. But when we account for the observed increase in the [PSI+] appearance rate in response to stress, we infer much higher overall selection in favor of [PSI+] modifiers, suggesting that [PSI+]-forming ability may be a consequence of selection for evolvability.THE yeast phenotype [PSI+] is characterized by prion aggregates of the protein Sup35. Cells are in either a [psi−] (normal) or [PSI+] state, depending on the absence or presence of the prion aggregates (Figure 1, a and b). Sup35 prion aggregates replicate in a similar fashion to mammalian prions but are cytoplasmic and, as such, the prion state is cytoplasmically inherited (Wickner et al. 1995).Open in a separate windowFigure 1.—Comparison between the three possible modes ([PSI+], genetic mimic, point mutation revertant) of the expression of 3′-UTR sequences in yeast. (a) The normal [psi−] phenotypic state; (b) the [PSI+] prion causes readthrough and low-level expression of 3′-UTRs across multiple genes, appearing at rate mPSI; (c) a genetic mimic of [PSI+] such as the sal3-4 mutant of Sup35 (Eaglestone et al. 1999) appearing at rate mmimic not reversible by the application of guanidine hydrochloride; (d) a point mutation in a single stop codon at rate μpoint, leading to incorporation of formerly 3′-UTR into a single coding sequence. (e) [PSI+] can act as a “stop-gap” mechanism, buying a lineage more time to acquire one or more adaptive stop codon readthrough point mutations. When this genetic assimilation is complete, [PSI+] can revert to [psi−] (Masel and Bergman 2003; Griswold and Masel 2009).When not part of an aggregate, Sup35 helps mediate translation termination in yeast (Stansfield et al. 1995b; Zhouravleva et al. 1995). Sup35 molecules that are incorporated into nonfunctional prion aggregates are presumably not available for translation termination, which can lead to the translation of stop codons by near-cognate tRNAs (Figure 1b) (Tuite and Mclaughlin 1982; Pure et al. 1985; Lin et al. 1986). This partial loss of Sup35 function leads to an increased frequency of readthrough translation of 3′-untranslated regions (3′-UTR) across all genes (Figure 1b). This increase is modest in wild-type yeast, from an average readthrough rate of 0.3% in [psi−] cells up to 1% in [PSI+] cells (Firoozan et al. 1991). Some [PSI+] yeast strains grow faster than [psi−] controls in certain harsh environments, suggesting that readthrough translation of some 3′-UTRs may be adaptive in certain conditions (True and Lindquist 2000; Joseph and Kirkpatrick 2008). This directly shows that [PSI+]-mediated capacitance may increase evolvability in the laboratory. [PSI+]-mediated phenotypes have a complex genetic basis, involving multiple loci (True et al. 2004).As an epigenetically inherited protein aggregate, [PSI+] can easily be lost after some generations (Cox et al. 1980). This returns the lineage to its normal [psi−] state and restores translation fidelity. If a subset of revealed phenotypic variation is adaptive, it may have lost its dependence on [PSI+] by this time (True et al. 2004). This process of genetic assimilation may, for example, involve one or more point mutations in stop codons, increasing readthrough up to 100% (Figure 1e) (Griswold and Masel 2009). This leaves the yeast with a new adaptive trait and with no permanent load of other, deleterious variation.In general, stop codons can be lost either directly through point mutations or indirectly through upstream indels. This leads to novel coding sequence coming from in-frame and out-of-frame 3′-UTRs, respectively. [PSI+] is expected to facilitate only the former, while mutation bias favors the latter. Yeasts show a much higher ratio of in-frame to out-of-frame 3′-UTR incorporation events than mammals do (Giacomelli et al. 2007), confirming a role for [PSI+] in capacitance-mediated evolvability in natural populations.The adaptive evolution both of evolvability in general (Sniegowski and Murphy 2006; Lynch 2007; Pigliucci 2008) and of capacitance in particular (Dickinson and Seger 1999; Wagner et al. 1999; Partridge and Barton 2000; Brookfield 2001; Pal 2001; Meiklejohn and Hartl 2002; Ruden et al. 2003) is highly controversial. In general, any costs of evolvability are borne in the present, while the benefits lie in the future, making it difficult for natural selection to favor an evolvability allele. For example, mutation rates seem to be set according to a trade-off between metabolic cost (favoring higher mutation rates) and the avoidance of deleterious effects (favoring lower mutation rates) (Sniegowski et al. 2000). The fact that mutation creates variation, the ultimate source of evolvability, is merely a fortuitous consequence of the metabolic cost of fidelity.Previous theoretical population genetic studies have, however, suggested that modifier alleles promoting the formation of [PSI+] might, unlike mutator alleles, be favored for their evolvability properties (King and Masel 2007; Masel et al. 2007; Griswold and Masel 2009; Masel and Griswold 2009). These models depend, however, on a number of parameter estimates. In particular, a number of predictions depend on the spontaneous rate of [PSI+] formation (Masel and Griswold 2009).
[PSI+] appearance rates and the fluctuation test:
The most widely cited spontaneous appearance rate for [PSI+] is mPSI ∼ 10−7–10−5, on the basis of experiments by Lund and Cox (1981). This estimate was calculated as the proportion of colonies scored as [PSI+] after growth over multiple generations from a single founding [psi−] clone. If [PSI+] happens to appear in the first generation of growth, this leads to a “jackpot” event with only one switching event, but many [PSI+] colonies. The proportion of colonies scored as [PSI+] therefore yields a systematic overestimation of the [PSI+] appearance rate.Various implementations of the fluctuation test (Luria and Delbrück 1943) can address such effects. The mutation rate experiment is replicated many times using independent populations, and a Luria–Delbrück distribution is fitted to the results across all replicates. In a simulation study, Stewart (1994) examined a number of estimators of the underlying Luria–Delbrück distribution and found that the maximum-likelihood estimator performed the best.Originally developed to study mutation rates, the fluctuation test can also be used for estimating epimutation rates. Fluctuation tests have been used to estimate the rate of gene silencing in Chinese hamster ovary cells (Holliday and Ho 1998) and in the yeast Schizosaccharomyces pombe (Singh and Klar 2002). However, fluctuation tests do not appear to be used routinely for epimutation rate estimates. For example, although the rates of spontaneous appearance and disappearance of [ISP+], a prion-like element in yeast, have been measured using the fluctuation test (Volkov et al. 2002), to the best of our knowledge there are no published estimates of the spontaneous rate of [PSI+] appearance as measured using a fluctuation test. Although results from the fluctuation test can be confounded by reverse epimutation, or back-switching, this is an issue only if the rate of back-switching is very high, e.g., 10−1–10−2 per generation (Saunders et al. 2003). This is not the case for [PSI+], for which the reverse epimutation rate (loss of [PSI+]) is <2 × 10−4 (Tank et al. 2007).Other [PSI+]-like phenotypes, including genetic mimics:
[PSI+] causes partial loss of Sup35 function, leading to elevated rates of translational readthrough at all stop codons (Figure 1b). There are many other spontaneous changes, presumably mutations, that also lead to elevated translational readthrough (Lund and Cox 1981). Mutations that affect readthrough at all stop codons (Figure 1c) (sometimes called “[PSI+]-like”) can be considered as genetic “mimics” because they produce the same phenotype as the Sup35 aggregate, but are generally not epigenetically inherited. A specific example of such a genetic mimic was characterized by Eaglestone et al. (1999), who identified the sal3-4 point mutation in the SUP35 gene. This leads to a defect in the Sup35 protein structure rendering the termination process less efficient (Eaglestone et al. 1999). The sal3-4 mutant can therefore be considered a partial loss-of-function genetic mimic of [PSI+], since it generates the same readthrough phenotype. Translation termination could also potentially be impaired through other point mutations or deletions, for example, in either the SUP35 or the SUP45 gene (Stansfield et al. 1995a) or in a tRNA that mutates to recognize stop codons at a higher rate. The presence of genetic mimics, whose effects are less reversible than those of [PSI+], can affect the evolution of the evolvability properties of the [PSI+] system such as its epimutation rate (Lancaster and Masel 2009). Note that genetic mimics are quite different from much rarer point mutations that convert stop codons into coding sequence (Figure 1d), resulting in readthrough at a single gene rather than multiple genes.Here we performed experiments to obtain accurate and precise estimates of the baseline appearance rates of both [PSI+] and [PSI+]-like phenotypes in permissive laboratory conditions, excluding stop codon point mutations that affect only a single gene. Our estimates are superior to previous estimates, since we use the fluctuation test. We consider the consequences of these estimates for the evolution of the [PSI+] system. 相似文献20.
Rearrangement of a 1,3-trans-[Pt(NH3)2[(GXG)-N7G,N7G]] intrastrand cross-link into interstrand cross-links within RNA duplexes
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The cross-linking reaction described previously in the DNA and 2′-O-methyl RNA series is extended to RNA duplexes. A 17mer single-stranded RNA containing the 1,3-trans-{Pt(NH3)2[(GAG)-N7G,N7G]} intrastrand chelate, named G*AG* (* indicating a platinated base) gives, upon pairing with the complementary RNA strand, the G*AG/CUC* interstrand cross-link. The rate of the reaction in 200 mM NaClO4 is similar to that observed for DNA–RNA duplexes. It depends on the added Na+ or Mg2+ cation and on its concentration. RNA duplexes containing GA/GA or AG/AG tandem mismatches in the rearrangement triplet core were also studied. The major interstrand cross-links, G*AG/CGA* and G*AG/AGC*, are accompanied by a minor one involving the central G of the CGA or AGC complementary sequence G*AG/CG*A and G*AG/AG*C. In 200 mM NaClO4, the G*A/GA tandem mismatch does not modify the rate of the cross-linking rearrangement whereas the AG*/AG mismatch slows it down by a factor of four. Our results reflect the predominance of the local structure of the rearrangement core over the nucleophility of the cross-linking base. They also show that the reaction could be used to trap tertiary structures of naturally occurring RNAs, including those with the commonly encountered GA/GA mismatch. 相似文献