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1.
The Drosophila Mos1 element can be mobilized in species ranging from prokaryotes to protozoans and vertebrates, and the purified transposase can be used for in vitro transposition assays. In this report we developed a ‘mini-Mos1’ element and describe a number of useful derivatives suitable for transposon mutagenesis in vivo or in vitro. Several of these allow the creation and/or selection of tripartite protein fusions to a green fluorescent protein–phleomycin resistance (GFP-PHLEO) reporter/selectable marker. Such X-GFP-PHLEO-X fusions have the advantage of retaining 5′ and 3′ regulatory information and N- and C-terminal protein targeting domains. A Mos1 derivative suitable for use in transposon-insertion mediated linker insertion (TIMLI) mutagenesis is described, and transposons bearing selectable markers suitable for use in the protozoan parasite Leishmania were made and tested. A novel ‘negative selection’ approach was developed which permits in vitro assays of transposons lacking bacterial selectable markers. Application of this assay to several Mos1 elements developed for use in insects suggests that the large mariner pM[cn] element used previously in vivo is poorly active in vitro, while the Mos1-Act-EGFP transposon is highly active.  相似文献   

2.
沈丹  陈才  王赛赛  陈伟  高波  宋成义 《遗传》2017,39(1):1-13
随着高通量测序技术的迅猛发展,越来越多的生物基因组注释结果表明:转座子几乎存在于所有生物的基因组中,是大多数生物基因组的重要组分。其中,Tc1/Mariner转座子是自然界中分布最广泛的一类DNA转座子超家族,在自然界已经发现14个有活性的Tc1/Mariner转座子(如Minos,Mos1等),另外通过分子重构也获得高活性的人工转座子,如睡美人转座子(Sleeping Beauty, SB)。SBMos1等转座子作为基因转移载体已被广泛应用于转基因、基因捕获和基因治疗等领域的研究中,并取得了很好的应用效果。本文将重点综述Tc1/Mariner转座子的结构、分类、分布、转座机制、活性转座子的挖掘,及其在转基因、基因捕获和基因治疗等研究领域的应用。  相似文献   

3.
4.
After intracellular in vitro exposure to the mutagenic and carcinogenic N-nitroso compounds N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), respectively, the average relative amounts of the premutational lesion O6-alkylguanine represent about 6% and 8% of all alkylation products formed in genomic DNA. At the level of individual DNA molecules gunine-O6 alkylation does nor occur at random; rather, the probability of a substitution reaction at the nucleophilic O6 atom is influenced by nucleotide sequence, DNA conformation, and chromatin structure. In the present study, 5 different double-stranded polydeoxynucleotides and 15 double-stranded oligodeoxynucleotides (24-mers) were reacted with MeNU or EtNU in vitro under standardized conditions. Using a competitive radioimmunoassay in conjunction with an anti-(O6-2′-deoxyguanosine) monoclonal antibody, the frequency of guanine-O6 alkylation was found to be strongly dependent on the nature of the nucleotides flanking guanine on the 5 and 3′ sides. Thus, a 5′ neighboring guanine, followed by 5 adenine and 5′ cytosine, provided an up to 10-fold more ‘permissive’ condition for O6-alkylation of the central guanine than a 5′ thymine (with a 5-methylcytocine in the 5′ position being only slightly less inhibitory). Thymine and cytosine were more ‘permissive’ when placed 3′ in comparison with their affects in the 5′ flanking position.  相似文献   

5.
《Gene》1998,222(2):213-222
Two artificial transposons have been constructed that carry a gene encoding Green Fluorescent Protein and can be used for generating libraries of GFP fusions in a gene of interest. One such element, AT2GFP, can be used to generate GFP insertions in frame with the amino acid sequence of the protein of interest, with a stop codon at the end of the GFP coding sequence; AT2GFP also contains a selectable marker that confers trimethoprim resistance in bacteria. The second element, GS, can be used to generate tribrid GFP fusions because there is no stop codon in the GFP transposon, and the resulting fusion proteins contain the entire amino acid sequence encoded by the gene. The GS element consists of a gfp open reading frame and a supF amber suppressor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open reading frame, and thus it can be translated continuously with the gfp amino acid sequence. As a target for GFP insertions, we used a plasmid carrying the native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resulting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Libraries of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular localization signals of proteins.  相似文献   

6.
The aminoglycoside antibiotic paromomycin that is highly toxic to the green alga Volvox carteri is efficiently inactivated by aminoglycoside 3′-phosphotransferase from Streptomyces rimosus. Therefore, we made constructs in which the bacterial aphH gene encoding this enzyme was combined with Volvox cis-regulatory elements in an attempt to develop a new dominant selectable marker – paromomycin resistance (PmR) – for use in Volvox nuclear transformation. The construct that provided the most efficient transformation was one in which aphH was placed between a chimeric promoter that was generated by fusing the Volvox hsp70 and rbcS3 promoters and the 3′ UTR of the Volvox rbcS3 gene. When this plasmid was used in combination with a high-impact biolistic device, the frequency of stable PmR transformants ranged about 15 per 106 target cells. Due to rapid and sharp selection, PmR transformants were readily isolated after six days, which is half the time required for previously used markers. Co-transformation of an unselected marker ranged about 30%. The chimeric aphH gene was stably integrated into the Volvox genome, frequently as tandem multiple copies, and was expressed at a level that made selection of PmR transformants simple and unambiguous. This makes the engineered bacterial aphH gene an efficient dominant selection marker for the transformation and co-transformation of a broad range of V. carteri strains without the recurring need for using auxotrophic recipient strains.  相似文献   

7.
Ehrlich KC  Montalbano BG  Cary JW 《Gene》1999,230(2):249-257
AFLR is a Zn2Cys6-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN5CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN5CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN5CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.  相似文献   

8.
The viscoelastic properties of aqueous solutions of the exocellular polysaccharide of Cyanospira capsulata have been studied, over a wide range of polymer concentrations, using small deformation oscillatory, steady and transient shear methods. The viscoelastic spectra generally resemble those of an entangled network, although notable deviations can be observed in the low frequency dependence of G′ and G″. At higher polymer concentrations, the viscoelastic spectrum shows solid-like behaviour over a wide range of frequencies. The superposition of η*(ω) and η( ) curves occurs only at low frequencies, at higher frequencies the slope of η*(ω) is lower than that of η( ). By studying the time evolution of shear stress after the inception of a steady shear rate (stress overshoot), the recovery of non-linear properties after steady shearing flow is seen to occur after times of c. 103 s (in the case of 1·1% w/v solutions).

The overall viscoelastic properties appear original in comparison with those of the two structurally limiting types of polysaccharide, the ‘ordered’ chain xanthan and the ‘random coil’ guar. A rationale for this ‘anomalous’ viscoelastic behaviour can be tentatively proposed in terms of flickering intermolecular cross-interactions between semi-flexible segments, which occur in addition to the usual topological constraints.  相似文献   


9.
Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments’ interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506–793) and CaDH2 (residues 683–767) on the structure of actin–tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin–tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle ‘ghost’ fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)–phalloidin or fluorescein-5′-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters ΦA, ΦE, Θ1/2 and N were calculated. ΦA and ΦE are angles between the fiber axis and the absorption and emission dipoles, respectively; Θ1/2 is the angle between the F-actin filament axis and the fiber axis; N is the relative number of randomly oriented fluorophores. Actin–tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the ‘on’ conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca2+) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca2+) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between ‘off’ (caldesmon and CaDH1) and ‘on’ (S-1 and CaDH2) states in a manner analogous to troponin.  相似文献   

10.
Mycobacterium tuberculosis and Mycobacterium avium infections cause the two most important mycobacterioses, leading to increased mortality in patients with AIDS. Various 5-substituted 2′-deoxyuridines, uridines, 2′-O-methyluridine, 2′-ribofluoro-2′-deoxyuridines, 3′-substituted-2′,3′-dideoxy uridines, 2′,3′-dideoxyuridines, and 2′,3′-didehydro-2′,3′-dideoxyuridines were synthesized and evaluated for their in vitro inhibitory activity against M. bovis and M. avium. 5-(C-1 Substituted)-2′-deoxyuridine derivatives emerged as potent inhibitors of M. avium (MIC90 = 1–5 μg/mL range). The nature of C-5 substituents in the 2′-deoxyuridine series appeared to be a determinant of anti-mycobacterial activity. This new class of inhibitors could serve as useful compounds for the design and study of new anti-tuberculosis agents.  相似文献   

11.
12.
Bark, wood and leaves of Ocotea catharinensis contain respectively 10 (average yield 0.7%.), 15 (average yield 0.004%.) and one (yield 0.4%.) neolignans of the bicyclo[3.2.1]octanoid and the hydrobenzofuranoid structural types, including the new rel-(7S,8R,1′R,4′S,5′R,6′R)-Δ8′-4′,6′-dihydroxy-5′-methoxy-3,4-methylenedioxy-3′-oxo-8.1′,7.5′-neolignan, (7S,8S)-Δ1′,3′,5′,8′-5,3′,5′-trimethoxy-3,4-methylenedioxy-8.1′,7.O.6′,4.O.7′-neolignan, (7R,8S,1′R,3′R)-Δ5′,8′-3,4,3′,5′-tetramethoxy-4′-oxo-8.1′,7.O.6′-neolignan and rel-(7R,8S,1′R,2′S)-Δ4′,8′-2′-hydroxy-3,4-dimethoxy-3′-oxo-8.1′,7.O.2′-neolignan.  相似文献   

13.
14.
We describe here an efficient synthesis of new 5-azaindolocarbazoles designed for cytotoxic and Chk1 inhibiting properties. The synthesis of ‘symmetrical’ and ‘dissymmetrical’ structures is discussed. Concerning the dissymmetrical 5-azaindolocarbazoles derivatives, with both an indole moiety and a 5-azaindole moiety, the synthesis was achieved using two very efficient key steps. The first one is a Stille reaction with a 3-trimethylstannyl-5-azaindole derivative and the second one a photochemical step leading to the proposed polycyclic structure. Various pharmacomodulations were performed to investigate the structure–activity relationships (SAR). Several substituents such as OBn, OH, and methylenedioxy groups were successfully introduced on the indole moiety of the 5-azaindolocarbazole. Compounds with or without substituents on the nitrogen atom of the maleimide were prepared, as well as derivatives with glucopyranosyl substituent on the nitrogen atom of the indole moiety. The cytotoxicity of these new compounds was evaluated on two cell lines (L1210, HT29). Several compounds showed cytotoxicity in the sub-micromolar range. Among the most cytototoxic was the 1,3-dioxolo[4,5-b]-6-(2-dimethylaminoethyl)-1H-pyrido[3′,4′:4,5]pyrrolo[3,2-i]pyrrolo[3,4-g]carbazole-5,7(6H,12H)-dione (35, IC50 = 195 nM on L1210). The compounds were also investigated for their Chk1 inhibiting activity. Compounds without any substitution on the maleimide moiety were the most potent. This is the case of compounds 45–47 with IC50 of, respectively, 72, 27, and 14 nM toward Chk1. Compound 46, which exhibits moderate cytotoxicity, appears to be a good candidate for development in a multi-drug anticancer therapy.  相似文献   

15.
Recent results are discussed which have led to a two-step model for UV mutagenesis in excision-deficient Escherichia coli. After exposure to UV, the replication fork is assumed to continue until immediately before certain photoproducts where it stops and leaves a gap which cannot be dealt with by recombination repair. In the first (misincorporation) step, bases (a proportion of which are ‘wrong’) are postulated to be inserted opposite the photoproduct under the direct influence of the recA gene product. These misincorporated bases can be revealed as mutations by delayed photoreversal in umuD, C and lexA (ind) bacteria. Their level is determined by the particular allele of recA that is present (recA441 > recA+ > recA430) and their rate of formation by the amount of recA protein in the cell and the degree of enrichment of the medium. No other protein needs to be synthesized for this step to occur. The second (bypass) step requires induced levels of the products of the umuD and C genes which are postulated to facilitate continued DNA synthesis on the priming end opposite the photoproduct. In principle, further errors could be made at this stage which might appear as ‘hitch-hiking’ rather than ‘targeted’ mutations.  相似文献   

16.
The SOS hypothesis postulated that the mutator effect on undameged DNA that generates phage-untargeted mutagenesis (UTM) results directly from the mechanism of targeted mutagenesis. RecA protein, which stimulates the cleavage of both the LexA repressor and UmuD protein, and the UmuDC gene products are required for UV-induced targeted mutagenesis. The use of phage λ for analyzing UV-induced mutagenesis has permitted a distinction to be made between the mechanisms of targeted and untargeted mutagenesis, in that the two processes differ with respect to their genetic requirements for recA+ and umuDC+ genes. In this paper, we show thet (i) proficiency for excision repair is required for UTM in double-stranded DNA phage but not in single-stranded DNA phage; (ii) the umuC function, which is not required for UTM of the double-stranded DNA phage λ, is necessary for untargeted mutagenesis of the single-stranded DNA phages M13 and φX174; (iii) for both single-stranded and double-stranded DNA phage, UV irradiation of the host increases the level of recA730-induced UTM. Our results are also consistent with the interpretation that the expression of untargeted mutagenesis in phage λ and in M13 depends on the polymerase and to a lesser extent on the exonuclease 5′ → 3′, activities of Po1I. These results suggest that the involvement of the RecA and UmuDC proteins may be related to more than the presence of base damage in the DNA substrate.  相似文献   

17.
Poplar cuttings of a resistant clone, Populus ‘Grandis’, and susceptible clones, Populus nigra ‘Italica’ and Populus ‘Robusta’, were infected with the pathogenic fungus Dothichiza populea alone, or with the pathogen and one of five strains of epiphytes antagonistic towards it (in vitro), isolated from poplar bark. The extent of injury was examined for 28 days after infection by determining the length of necrotic patches and their area as expressed in per cent of the total area of a cutting or the area of necrotic injuries caused by the pathogen alone.

All the poplar cuttings of both the resistant and susceptible clones became diseased when infected with the pathogen alone. Surprisingly enough, however, the least affected clone was the susceptible P. ‘Robusta’, in which necrotic injuries covered 28% of the total area, as against 40% and 70% in the resistant P. ‘Grandis’ and the susceptible P. nigra ‘Italica’, respectively.

When the cuttings were infected simultaneously with Dothichiza populea and its antagonistic epiphytes, the diseased area in the resistant clone diminished by as much as two-thirds, and in the susceptible P. nigra ‘Italica’, by one-third in comparison with the area affected by the pathogen alone. In turn, in the susceptible P. ‘Robusta’ the introduction of three out of five epiphytes stimulated the growth of the pathogenic fungus producing on average a double increase in the necrotic area. The differences in the response of the pathogen to the presence of epiphytes recorded in the susceptible clones indicate a marked influence of the plant on the nature of interactions between its epiphytic microflora and the pathogen.  相似文献   


18.
Brittle rachis is an important trait to elucidate the domestication process in barley. Brittle rachis in wild barley (Hordeum vulgare ssp. spontaneum (C. Koch.) Thell) is controlled by two dominant complementary genes, Btr1 and Btr2. Cultivated barley (H. vulgare L. ssp. vulgare) lost the brittle rachis character during domestication as a result of mutation at the Btr1 or Btr2 locus. In this study, a high-resolution map of the btr1 locus was constructed using an F2 population of cultivar (cv. ‘Kanto Nakate Gold’) × wild barley (line OUH602). We cloned and sequenced 26 AFLP markers linked with the btr1 and btr2 loci. Ten converted STS markers were located on the short arm of chromosome 3H only, and at least 9 of the 10 STS markers were allelic with their original AFLPs. Efficient conversion of co-dominant STS markers using BAC clones was successful. No suppression of recombination was observed in the btr1 region even though wild barley was used as one of the parents. Initial results of BAC screening confirmed the resolution power of the developed high resolution map.  相似文献   

19.
UV mutagenesis in E. coli is believed to occur in two discrete steps. The second step involves continued DNA synthesis beyond a blocking lesion in the template strand. This bypass step requires induced levels of umuD and umuC gene products and activated recA protein. DNA polymerase III may be involved since a dnaE mutator strain (believed to have defective base selection) is associated with enhanced UV mutagenesis in conjunction with a genetic background permitting the bypass step. In non-UV-mutable umu and lexA strains, UV mutagenesis can be demonstrated if delayed photorevesal is given. This is interpreted as indicating that an earlier misincorporation step can occur in such strains but the resulting mutations do not survive because the bypass step is blocked. The misincorporation step does not require any induced SOS gene products and can occur either at the replication fork or during repair replication following excision of a DNA lesion. Neither a dnaE mutator gene (leading to a defective subunit of DNA polymerase III holoenzyme) nor a mutD5 mutator gene (leading to a defective ε proofreading subunit) had any effect on he misincorporation step. Although this is consistent with DNA polymerase III holoenzyme not being involved in the misincorporation step, other interpretations involving the inhibition of ε proofreading activity by recA protein are possible.

In vitro studies are reported in which sites of termination of synthesis by DNA polymerase III holoenzyme on UV-irradiated M13 mp8 DNA were examined in the presence of inhibitors of the 3′–5′ proofreading exonuclease (including recA protein). No evidence was found for incorporation of bases opposite photoproducts suggesting that either inhibition is more complete in the cell and/or that other factors are involved in the misincorporation step.  相似文献   


20.
The roles of metals in the phosphodiester bond cleavage reaction performed by the hammerhead ribozyme are under investigation. In this study, the apparent affinities and the abilities of several different metals to support ribozyme activity are reported. The relative affinities of divalent cations for the hammerhead ribozyme are determined by measuring their ability to release bound Mn2+. The EPR-detected Mn2+ competition studies give an order of apparent affinity of Mn2+ Co2+ Zn2+>Cd2+Mg2+. This ordering generally follows the trend of maximum rates of cleavage determined at pH 7.0, 0.1 M NaCl, and saturating metal concentrations, of Mn2+>Co2+>Cd2+>Mg2+. The maximum rate is observed for Mn2+ under these conditions and may be related to the high affinity, low pKa and low ΔHhyd of this ion. Substitution of phosphorothioates 5′ to each of the nine adenosines in the enzyme strand yields a change in the Mn2+ binding properties of the hammerhead complex. In the phosphorothioate-substituted hammerhead complex, eight to nine Mn2+ bind in two types of classes: ‘type 1’ (n=1±0.3, Kd=1.1±1 μM) and weaker ‘type 2’ (n=7.7±0.3, Kd=125±27 μM). The multiple phosphorothioate substitutions result in the loss of two to three of the higher affinity sites observed in the unmodified ribozyme. Metal competition studies with the phosphorothioate-substituted ribozyme indicate that the relative affinities of the metals are Cd2+>Zn2+>Co2+, Mg2+ with the number of Mn2+ displaced and apparent affinity of the thiophilic Cd2+ most affected by the phosphorothioate substitutions.  相似文献   

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