Nucleotide sequence of the transposable element IS15

We have determined the complete nucleotide sequence of the right (R) copy of the insertion sequence IS15 which flanks, in direct orientation, the composite transposon Tn1525. IS15-R, which is capable of independent transposition, is 1648 bp long and has short (14 bp) perfect inverted repeats at its termini. Analysis of the nucleotide sequence indicates that IS15-R results from the transposition, in direct orientation, of a smaller (820 bp long) IS, designated IS15-Δ, into itself. This integration event is accompanied by the duplication of 8 bp in the target DNA. IS15-Δ possesses two large overlapping open reading frames (ORF) located on opposite strands. Because of this particular structure, IS15 possesses four large ORFs which, due to the integration event, exhibit some differences with those of the parental 1S15-Δ.     

The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66

A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn1721. A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn1721 and the new derivative, Tn5491, introduced onto a conjugative F' plasmid. The target sequence is inserted between two inverted resolution sites (res) of Tn1721 present on the high-copy plasmid pJOE2114. Due to the inviability of long palindromic sequences in Escherichia coli insertions between the inversely orientated res sites of pJOE2114 are positively selected. Transposition of Tn5491 into the target sequence is selected by cointegrate formation of Tn5491 during transposition, mating and transfer of the nonconjugative sequencing vector. After cointegrate resolution, the additional res sites in the vector result in a second site-specific recombination removing most of the transposon (except of 136 bp) and part of the target sequence. The reduced plasmid sizes and the use of the universal primer improved the quality of the sequencing results obtained on an automated fluorescent sequencer. A 3.35-kb EcoRI fragment from the 30-kb terminal inverted repeats (TIR) of the Streptomyces lividans chromosome was sequenced by this method. A 1304-bp sequence was found on this fragment with the features of insertion elements. The element called IS1372 had 27-bp IR and two potential open reading frames. The predicted gene products had similar sizes and high similarity to gene products encoded by insertion sequences of the IS3 family. Furthermore, a potential signal stimulating ribosomal shifts and typical for members of the IS3 family was identified. Five to seven copies of IS1372 were found in different strains of S. lividans but none in other Streptomyces species tested     

IS-like element IS8 in RP4 plasmid and its involvement in cointegration

The structure of the cointegrate plasmids formed by fusion of RP4 and the tumour-inducing plasmid (pTi) of Agrobacterium tumefaciens was analyzed. In all of the nine independently isolated pTi: :RP4 cointegrates, the integration occurred at the same site on the RP4 genome. Moreover, a 1.2 Md (1750 bp) RP4 sequence (IS8) was directly repeated at both junction sites of the two replicons. The insertion of RP4 generated deletions, starting from the IS8sequence and extending into the Ti part of the cointegrate. Dissociation of the cointegrates resulted in wild-type RP4 and Ti-plasmids with the IS8 sequence inserted at the original RP4 insertion site. The processes of integration and dissociation and the genetic properties of the cointegrates indicate that the IS8 sequence has unique characteristics defining a new insertion sequence.     

Transposition and targeting of the prokaryotic mobile element IS30 in zebrafish

We provide evidence that a prokaryotic insertion sequence (IS) element is active in a vertebrate system. The transposase of Escherichia coli element IS30 catalyzes both excision and integration in extrachromosomal DNA in zebrafish embryos. The transposase has a pronounced target preference, which is shown to be modified by fusing the enzyme to unrelated DNA binding proteins. Joining the transposase to the cI repressor of phage λ causes transposition primarily into the vicinity of the λ operator in E. coli, and linking to the DNA binding domain of Gli1 also directs the recombination activity of transposase near to the Gli1 binding site in zebrafish. Our results demonstrate the possibility of fusion transposases to acquire novel target specificity in both prokaryotes and eukaryotes.     

Two non-reactive ternary complexes of estrogenic 17beta-hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis.

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17β-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41).

To further study the transition state, two non-reactive ternary complexes, 17β-HSD1–EM519-NADP⁺ and 17β-HSD1–EM553-NADP⁺ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a=155.59 Å, b=42.82 Å, c=121.15 Å, β=128.5° for 17β-HSD1–EM519-NADP⁺, and a=124.01 Å, b=45.16 Å, c=61.40 Å, β=99.2° for 17β-HSD1–EM553-NADP⁺, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.     

Sequence of a canine cDNA clone encoding a Ran/TC4 GTP-binding protein

We report the isolation and characterization of a canine cDNA encoding a 216-amino acid GTP-binding protein of the Ras superfamily. The protein is almost identical to the human TC4 [Drivas et al., Mol. Cell. Biol. 10 (1990) 1793–1798] and Ran [Bischoffand Ponstingi, Proc. Natl. Acad. Sci. USA 88 (1991) 10830–10834; Nature 354 (1991) 80–82] proteins, the latter of which has been found to be involved in cell cycle control. Furthermore, the protein is highly similar to the fission yeast spi1 gene product [Matsumoto and Beach, Cell 66 (1991) 347–360]. The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.     

The flagellates of the termite Hodotermopsis sjoestedti: Immunological and ultrastructural characterization of four new species in the genera Spirotrichonympha, Spironympha and Microjoenia

Four species of spirotrichonymphids representing three genera Spirotrichonympha, Spironympha and Microjoenia symbiotic in the termite Hodotermopsis sjoestedti, have been studied by light and immunofluorescence microscopy and transmission electron microscopy. The genus Spirotrichonympha represented by Spirotrichonympha cincta n. sp. is characterized by a compound axostyle composed of several fibers or subaxostyles, and this species has peculiar undescribed structures associated with the flagellar lines. The genus Spironympha is characterized by flagellar lines restricted to the anterior area and a simple tubular axostyle limited by 1–3 layers of microtubules. The two new Spironympha species Spironympha obtusa and Spironympha oblonga are distinguished by peculiarities in the flagellar lines and the axostyle, as revealed by immunofluorescence and electron microscopy. The genus Microjoenia represented by Microjoenia minuta n. sp. has radiating flagellar lines and an axostyle with two microtubular rows originating from the pelta-axostyle rows covering the anterior cap. Four new species were named and one was assigned to the SSU rRNA sequences provided by the molecular phylogenetic studies by Ohkuma et al. [2000. Phylogenetic identification of hypermastigotes, Pseudotrichonympha, Spirotrichonympha, Holomastigotes, and parabasalian symbionts in the hindgut of termites. J. Eukaryot. Microbiol. 47, 249–259; 2005. Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences, with emphasis on the Hypermastigea. Mol. Phylogenet. Evol. 35, 646–655].     

Opsin biosynthesis and trans-cis isomerization of aldehyde form chromophore in the blowfly Calliphora erythrocephala eye

The effect of eye carotenoid content, light conditions and retinoid supply on the biosynthesis of opsin, as well as the ability to isomerize of exogenous all-trans-retinal to 11-cis-retinal were investigated in the photoreceptors of blowfly Calliphora. SDS-PAGE of digitonin extracts from isolated rhabdom fractions of carotenoid-fortified and carotenoid-deficient animals revealed, on heavily loaded gels, that in both cases opsin forms faint minor bands similar to the patterns of “R⁻-flies” described as “vitamin A-deficient flies” (Paulsen and Schwemer, Biochim. biophys Acta 557, 358–390, 1979) or “carotenoid-deficient flies” (Paulsen and Schwemer, Eur. J. Biochem. 137, 609–614, 1983. Similar opsin patterns were obtained in flies subjected to continuous 72 h blue or yellow-green light or to a 12 h: 12 h white light:dark cycle, or total darkness, irrespective of the carotenoid content in their eyes. 11-cis-retinal and, to a lesser extent, all-trans-retinal, when included in the diet or painted on the cornea, were found to stimulate opsin biosynthesis in the dark. 11-cis-retinal in the dark or all-trans-retinal after illumination of the flies with blue light were the most effective, as compared with the effect of all-trans-retinal in the dark. Exogenous all-trans-retinal in the dark can be partially converted into a mixture of 11-cis-retinal (26%) and 13-cis-retinal (74%) by fly retina homogenate in vitro. It was concluded that Calliphora opsin biosynthesis is not strongly dependent on the carotenoid supply or on light: dark conditions and is triggered by the 11-cis aldehyde form of the chromophore, which can be produced in the fly retina either by an isomerase system in the dark or as a result of photoisomerization.     

Redox potentials of the pyridine-haem c system

1. Haem c was synthesized and purified. It was shown unequivocally that the method gives a product with the cysteine residues on the -carbon atoms at the 2 and 4 positions of the haem.

2. Redox potentials of haem c in the presence of 2.5 M pyridine were determined in the pH range 1.5–13; it was found necessary to add cetyl trimethyl ammonium bromide (CTAB) to prevent precipitation in the acid range below about pH 4. The E_m vs pH curve shows three slopes (−dE/dpH) of value, 0.18, 0.01 and 0.06 with points of inflexion at pH 3.8 and 10.6. The potentials are intermediate between those of protohaem and mesohaem obtained under similar conditions.

3. With constant haem c concentration (a) 10⁻⁴ M and (b) 10⁻⁵ M and varying pyridine concentration (0.12–5 M) it was found at pH 9.0 that E_m values increased as the pyridine concentration was increased and there was a tendency to reach a plateau value. The explanation appears to be that pyridine binds more firmly to ferroporphyrin c than to ferriporhyrin c.

4. When the pyridine concentration was kept constant (2.5 M) and the haem c concentration was varied in the range 7 · 10⁻⁴–7 · 10⁻⁶ M, it was found that a decrease in haem c concentration brought about an increase in redox potential. The results are explained as being due to dimerization of the oxidized form.

5. The results are discussed in comparison with a number of related haem systems.     

CRISPR/Cas系统与志贺菌毒力和耐药的关系及插入序列IS600对cse2表达水平的影响北大核心CSCD

【目的】了解志贺菌中成簇的规律间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)的分布及其与毒力和耐药的关系,并分析志贺菌中插入序列IS600对CRISPR相关蛋白基因cse2 m RNA表达水平的影响。【方法】利用课题组前期设计的引物PCR扩增志贺菌的3个CRISPR位点、CRISPR相关蛋白基因cse2、耐药基因和毒力基因;改良Kirby-Bauer(K-B)纸片法进行药敏试验;台盼蓝计数试验检测细菌毒力;Real-time PCR检测志贺菌中cse2基因m RNA表达水平。分别分析志贺菌中CRISPR/Cas系统与耐药基因、耐药表型、毒力基因、毒力表型的关系;了解IS600对CRISPR相关蛋白基因cse2 m RNA表达水平的影响。【结果】志贺菌中CRISPR1位点阴性细菌的毒力强;插入序列IS600使cse2 m RNA表达水平降低。【结论】志贺菌中存在CRISPR1、2、3位点;CRISPR1位点与毒力有关;插入序列IS600对cse2 m RNA表达水平有影响。     

Escherichia coli leucine-responsive regulatory protein (Lrp) controls lysyl-tRNA synthetase expression

Using random Tn10 insertion mutagenesis, we isolated an Escherichia coli mutant strain affected in the regulation of lysU, the gene encoding the inducible form of lysyl-tRNA synthetase. The transposon giving rise to the altered expression of lysU was found inserted within lrp. The latter gene codes for the leucine-responsive regulatory protein (Lrp) which mediates a global response of the bacterium to leucine. An involvement of Lrp in the regulation of lysU was searched for by using a lysU-lacZ operon fusion. The following conclusions were reached: (i) inactivation of lrp causes an increased activity of the lysU promoter, whatever the growth conditions assayed, (ii) insertion of a wild-type lrp gene into a multi-copy plasmid significantly reduces lysU expression, and (iii) sensitivity of the lysU promoter to the presence of leucine in the growth medium is abolished in the lrp context.     

Trapping, loss and annihilation of excitations in a photosynthetic system: II. Experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata

In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, k_h, and the rate of trapping by an open reaction center, k^o_t, can be estimated. For R. rubrum it is found that λ = 14−17, k_h = (1−2)·10¹² s⁻¹ and k^o_t = (4−6)·10¹¹ s⁻¹, for Rps. capsulata λ ≈ 30, k_h ≈ 4·10¹¹ s⁻¹ and k^o_t ≈ 3·10¹¹ s⁻¹. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria.     

Protein-protein interactions of the light-harvesting chlorophyll a/b protein. II. Evidence for two stages of cation independent association

In a previous paper, we observed a two-stage cation-independent association of the light-harvesting chlorophyll a/b protein from spinach chloroplasts based on concentration-dependent changes in the sedimentation coefficient. The two stages of association occurred between (2–4) and (4–7) μg/ml chlorophyll. In this paper, we provide further evidence for this association.

This includes: (1) A decrease in the number of divalent cation binding sites in the second stage of association. (2) A corresponding decrease in the extent of the cation-dependent association. (3) A positive deviation from Beer's law for chlorophyll b for both stages of the cation-independent association and a positive deviation for chlorophyll a for the second stage of association only. (4) A change in the fluorescence emission of both chlorophyll a and b. The change for chlorophyll b was observed for both steps of association whereas that for chlorophyll a was observed for the second step of association only. Therefore, the first stage of association affects only chlorophyll b whereas the second stage alters the environment of both chlorophyll a and b. (5) In addition, divalent cations quenched chlorophyll fluorescence. However, the quenching which required 200–300 μM divalent cation for half-maximal effects was related neither to divalent cation binding nor to the divalent cation-induced association of the protein.     

尖孢镰刀菌亚麻专化型Folprp4基因参与调控菌丝生长和分生孢子发生

目的: 通过对尖孢镰刀菌中Folprp4基因的鉴定,揭示其在尖孢镰刀菌中的功能及致病相关性。方法: 基于同源重组原理,根据测定出的Folprp4基因序列,应用Split-Marker重组技术构建含有潮霉素抗性基因(hph)的基因缺失盒。将基因缺失盒经PEG介导转化到野生型原生质体中,在含有潮霉素B的TCC培养基上筛选转化子,通过PCR正负筛查获得Folprp4基因缺失突变株(ΔFolprp4)。构建含有Folprp4基因的载体pZDH1,并将其转化到敲除突变体中进行互补测验。结果: 与野生型(hm)和异位插入突变体(ecFolprp4)相比,敲除突变体菌丝生长受到严重阻碍,当野生型和异位插入突变体长满整个平板时,敲除突变体菌落呈小点状。敲除突变体的另一个显著变化是ΔFolprp4的分生孢子产量显著下降。侵染实验表明,ΔFolprp4对亚麻幼苗的毒力显著降低。互补实验表明,该互补载体的回复子(Folprp4-C)在菌落形态、生长速率、分生孢子产量和毒力方面均恢复到了野生型菌株。结论: Folprp4基因与尖孢镰刀菌的菌丝生长、分生孢子发生和致病性有关。     

The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece

The examination of macrophyte, water and sediment samples, collected at depths less than 1.5 m from 50 different sites along the North Aegean coasts, has revealed, for the first time in Greek coastal waters, the presence of two Ostreopsis species (O. ovata and O. cf. siamensis) and Coolia monotis in the majority of the sampling sites (94% and 100%, respectively). Other epiphytic dinoflagellates of the genera Prorocentrum and Amphidinium and diatoms were accompanying species in this epiphytic community. Morphometric features, plate formula and thecal ornamentation were used for species identification. O. ovata cells were smaller in dorsoventral (DV) diameter and width (W) (26.18–61.88 μm and 13.09–47.60 μm, respectively) in comparison with O. cf. siamensis (35.70–65.45 μm and 23.80–49.98 μm, respectively). In contrast, the anterioposterior (AP) diameter of O. cf. siamensis was smaller (14.28–26.18 μm) resulting in DV/AP ≈ 3, whereas the above ratio for O. ovata was less than 2 (AP ranging between 14.28–35.70 μm). Moreover, the theca of O. ovata cells was ornamented with scattered pores, which fluctuated in a wider range (0.07–0.32 μm) than those of O. cf. siamensis (0.23–0.29 μm). Coolia monotis cells were almost round with average DV diameter 26.88 μm, AP 25.66 μm and width 26.76 μm. Small and large cells were recorded in both field and culture populations of Ostreopsis spp. and C. monotis, while hyaline cysts were observed for O. ovata. The presence of O. ovata and O. cf. siamensis exhibited a clear seasonal pattern dominating (maximum abundance up to 4.05 × 10⁵ cells gr⁻¹ fwm) the period from midsummer to late autumn in years 2003 and 2004, while C. monotis was found also in winter and spring months.     

The distance between cytochromes a and a3 in the azide compound of bovine-heart cytochrome oxidase

The electron-spin relaxation rates of the two species of cytochrome a³⁺₃-azide found in the azide compound of bovine-heart cytochrome oxidase were measured by progressive microwave saturation at T = 10 K. It has been shown previously that Cyt a⁺³₃-azide gives rise to two distinct EPR resonances, depending upon the oxidation state of Cyt a. When Cyt a is ferrous, Cyt a³⁺₃-azide has g = 2.88, 2.19 and 1.64; upon oxidation of Cyt a, the a³⁺₃-azide g-values become g = 2.77, 2.18, and 1.74 (Goodman, G. (1984) J. Biol. Chem. 259, 15094–15099). The relaxation effect of Cyt a on Cyt a₃ could be measured as the difference in microwave field saturation parameter H_1/2 between the g = 2.77 and g = 2.88 species. For each signal the spin-lattice relaxation time T₁ was determined from H_1/2 using the transverse relaxation time T₂. The value of T₂ at 10 K was extrapolated from a plot of line-width vs. temperature at higher temperature. The dipolar contribution to T₁ was related to the Cyt a-Cyt a₃ spin-spin distance utilizing available information on the relative orientation of Cyt a₃-azide and Cyt a (Erecinska, M., Wilson, D.F. and Blasie, J.K. (1979) Biochim. Biophys. Acta 545, 352–364). By taking into account the relaxation parameters for both g_x and g_z components of the Cyt a₃-azide g-tensor, the angle between the g_z components of the Cyt a and Cyt a₃g-tensors was determined to be between 0 and 18°, and the Cyt a-Cyt a₃ spin-spin distance was found to be 19 ± 8 Å.     

In vitro excystment of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae)

1986. In vitro excystrnent of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae). International Journal for Parasitology 16: 641–645. An optimal hatching success of Plagiorchis species 1 metacercariae (100% excystment, active metacercariae, mean hatching speed 2–10 min, lowest variance of the mean speed) was observed after pretreatment in an HCl-pepsin solution at pH 2.0 and 42°C for 60–70 min, and incubation in a hatching medium at 42 °C and pH 7.3–8.0 with a bile salt (Nacholate), NaHCO₃, and a reductant (cysteine with 100% N₂). The minimum conditions for nearly 100% excystment with lower hatching speeds and higher variances were the presence of NaHCO₃, an oxygen concentration reduced to about 3% in the gas phase, pH> 7.3 and a temperature near 30°C if Na-cholate was absent, or in the presence of the bile salt, a phosphate buffer at pH> 5.0 and room temperature only. Obviously some hatching factors acted interchangeably with compensation for missing stimuli by others. The effect of the bile salt was comparable with that of other surfactants. The metacercariae excysted in nonenzymatic media, which implies an active hatching mechanism.