一个RNAi载体上反向重复片段的测序策略

相邻的反向重复DNA片段有形成单链内二级结构的倾向,属于一种测序困难的DNA模板。解决RNAi载体插入的反向重复片段的测序问题,为该类载体正确性的测序验证奠定基础。采用常规分子克隆方法构建表达小麦TaATG2串联反向重复片段的RNAi载体,设计2种策略对经菌落PCR初步鉴定的载体进行测序验证:一种是以完整的载体质粒为模板进行测序;另一种是先对载体进行酶切处理,切除反向重复片段中的一个后对保留另一个片段的线性载体进行测序。结果表明,第一种测序策略受到串联反向重复片段形成的单链内部二级结构的影响,测序信号在反向重复片段处出现衰减或乱峰,无法读取序列。第二种测序策略排除了2个反向重复片段之间的干扰,保留在载体上的片段测序信号清晰,序列准确。采用酶切切除一个片段后进行测序的方法,经过2次酶切和2次测序可以有效地对载体上的2个反向重复片段分别进行序列测定,进而确认构建载体的正确性。     

Alternate Junctions and Microheterogeneity of Tlr1, a Developmentally Regulated DNA Rearrangement in Tetrahymena thermophila

ABSTRACT. A large number of developmentally regulated DNA rearrangements occur during the development of the macronucleus in Tetrahymena thermophila , Tlr1 is a deletion element which has large inverted repeats near the rearrangement junctions and deletes more than 13 kbp of internal DNA. Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 rearrangement in B strain cells. We show here that C2 strain Tetrahymena also use alternate rearrangement junctions. We have mapped and sequenced two additional rearrangement variants and find that both the left and right can vary over a range of approximately 200 bp. We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 rearrangement product.     

How a Kantian Can Accept Evolutionary Metaethics

Contrary to widely held assumptions, an evolutionary metaethics need not be non-cognitivist. I define evolutionary metaethics as the claim that certain phenotypic traits expressing certain genes are both necessary and sufficient for explanation of all other phenotypic traits we consider morally significant. A review of the influential cognitivist Immanuel Kants metaethics shows that much of his ethical theory is independent of the anti-naturalist metaphysics of transcendental idealism which itself is incompatible with evolutionary metaethics. By matching those independent aspects to an evolutionary metaethics a cognitivist Kantian evolutionary metaethical theory is a possibility for researchers to consider.     

Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression

Analysis of Agrobacterium-transferred DNA (T-DNA) revealed strong correlations between transgene structures and floral pigmentation patterns from chalcone synthase (chs) co-suppression among 47 Petunia transformants. Presented here are the full details of T-DNA structural organization in that population. Sixteen transformants (34%) carried one T-DNA copy while 31 (66%) carried 106 complete and partial T-DNA elements in 54 linkage groups. Thirty linkage groups contained multiple T-DNA copies; 15 of these contained only contiguously repeated copies, 8 contained only dispersed copies and 7 contained both. Right-border inverted repeats were three times more frequent than left-border inverted or direct repeats. Large fragments of binary-vector sequences were linked to the T-DNA in seven plants.     

How Microbes Can Achieve Balanced Growth in a Fluctuating Environment

A microbial colony needs several essential nutrients in order to grow. Moreover, the colony requires these nutrients in fixed combinations, which are dictated by the chemical composition of its biomass. Unfortunately, ambient availabilities of the various nutrients vary all the time. This poses the question of how microbes can achieve balanced growth.The present solution to this problem is novel in that the allocation of molecular building blocks among assimilatory machineries within the cell is regarded as dynamic. This paper shows that allocation can be adapted so as to achieve balanced growth, nearly regardless of environmental conditions. Moreover, it is shown that a feedback mechanism, which monitors internal stores, is able to achieve this allocation.     

The bz-rcy allele of the Cy transposable element system of Zea mays contains a Mu-like element insertion

Summary The receptive component of theCy transposable element system (rcy: Mu7) at theBz locus ofZea mays L. is 2.2 kb and has long terminal inverted repeats. The insertion is flanked by a 9 bp duplication. In the presence of an autonomousCy element in the genome,rcy: Mu7 is excised frombz-rcy in a manner consistent with a model suggested previously. The termini ofrcy: Mu7 have 85% sequence similarity with theMu1 element ofZ. mays. This is consistent with the observation thatMu1 can behave genetically like a receptive component of theCy system.     

Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).     

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     

Immobilized copies with a nearly intact structure of the transposon Tam3 in Antirrhinum majus: implications for the cis-element related to the transposition

 In this study we have focused on two copies of the transposon Tam3 isolated from an Antirrhinum majus plant which has flower variegation due to the excision of Tam3 from the nivea locus. These two copies possess a high homology, over 95%, to an active Tam3 element found in the nivea ^{recurrence:Tam3} allele. Although somatic excision of the Tam3 copy from the nivea locus can be detected at 15°C by Southern blotting, neither of the two copies showed any sign of the excision. Both of the immobilized copies were also found in five varieties from different A. majus sources, all of which contain common fragments. The results suggest that the two copies have been fixed in the genomes of many A. majus varieties. Structural differences between these immobilized copies and the known active copy were mainly observed in the subterminal regions, including the terminal inverted repeats. The immobility of the two Tam3 copies might be due to mutations within the end regions of essential cis-elements in Tam3 transposition, as reported for Ac and En/Spm. Received: 30 June 1997 / Accepted: 5 August 1997     

How Can the Eco‐efficiency of a Region be Measured and Monitored?

The concept of eco-efficiency is commonly referred to as a business link to sustainable development. In this article, ecoefficiency is examined at a regional level as an approach to promoting the competitiveness of economic activities in the Finnish Kymenlaakso region and mitigating their harmful impacts on the environment. The aim is to develop appropriate indicators for monitoring changes in the eco-efficiency of the region. A starting point is to produce indicators for the environmental and economic dimensions of regional development and use them for measuring regional eco-efficiency. The environmental impact indicators are based on a life-cycle assessment method, producing different types of environmental impact indicators: pressure indicators (e.g., emissions of CO₂), impact category indicators (e.g., CO₂ equivalents in the case of climate change), and a total impact indicator (aggregating different impact category indicator results into a single value). Environmental impact indicators based on direct material input, total material input, and total material requirement of the Kymenlaakso region are also assessed. The economic indicators used are the gross domestic product, the value added, and the output of the main economic sectors of Kymenlaakso. In the eco-efficiency assessment, the economic and environmental impact indicators are monitored in the same graph. In a few cases eco-efficiency ratios can also be calculated (the economic indicators are divided by the environmental indicators). Output (= value added + intermediate consumption) is used as an economic indicator related to the environmental impact indicators, which also cover the upstream processes of the region's activities. In the article, we also discuss the strengths and weaknesses of using the different environmental impact indicators.     

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli

Summary The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).     

Transposase-transposase interactions in MOS1 complexes: a biochemical approach

Transposases are proteins that have assumed the mobility of class II transposable elements. In order to map the interfaces involved in transposase-transposase interactions, we have taken advantage of 12 transposase mutants that impair mariner transposase-transposase interactions taking place during transposition. Our data indicate that transposase-transposase interactions regulating Mos1 transposition are sophisticated and result from (i) active MOS1 dimerization through the first HTH of the N-terminal domain, which leads to inverted terminal repeat (ITR) binding; (ii) inactive dimerization carried by part of the C-terminal domain, which prevents ITR binding; and (iii) oligomerization. Inactive dimers are nonpermissive in organizing complexes that produce ITR binding, but the interfaces (or interactions) supplied in this state could play a role in the various rearrangements needed during transposition. Oligomerization is probably not due to a specific MOS1 domain, but rather the result of nonspecific interactions resulting from incorrect folding of the protein. Our data also suggest that the MOS1 catalytic domain is a main actor in the overall organization of MOS1, thus playing a role in MOS1 oligomerization. Finally, we propose that MOS1 behaves as predicted by the pre-equilibrium existing model, whereby proteins are found to exist simultaneously in populations with diverse conformations, monomers and active and inactive dimers for MOS1. We were able to identify several MOS1 mutants that modify this pre-existing equilibrium. According to their properties, some of these mutants will be useful tools to break down the remaining gaps in our understanding of mariner transposition.     

Low-Resolution Density Maps from Atomic Models: How Stepping “Back” Can Be a Step “Forward”

Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15–40 Å), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction.     

FB-NOF is a non-autonomous transposable element,expressed in Drosophila melanogaster and present only in the melanogaster group

Most foldback elements are defective due to the lack of coding sequences but some are associated with coding sequences and may represent the entire element. This is the case of the NOF sequences found in the FB of Drosophila melanogaster, formerly considered as an autonomous TE and currently proposed as part of the so-called FB-NOF element, the transposon that would be complete and fully functional. NOF is always associated with FB and never seen apart from the FB inverted repeats (IR). This is the reason why the FB-NOF composite element can be considered the complete element. At least one of its ORFs encodes a protein that has always been considered its transposase, but no detailed studies have been carried out to verify this.     

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 22 associated with cat eye syndrome

We present prenatal diagnosis of mosaicism for a small supernumerary marker chromosome (sSMC) derived from chromosome 22 associated with cat eye syndrome (CES) using cultured amniocytes in a pregnancy with fetal microcephaly, intrauterine growth restriction, left renal hypoplasia, total anomalous pulmonary venous return with dominant right heart and right ear deformity. The sSMC was bisatellited and dicentric, and was characterized by multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH). The SALSA MLPA P250-B1 DiGeorge Probemix showed duplication of gene dosage in the CES region. aCGH showed a 1.26-Mb duplication at 22q11.1–q11.21 encompassing CECR1–CECR7. The sSMC was likely inv dup(22) (q11.21). Prenatal diagnosis of an sSMC(22) at amniocentesis should alert CES. MLPA, aCGH and fetal ultrasound are useful for rapid diagnosis of CES in case of prenatally detected sSMC(22).     

Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microheterogenous cytochrome P-450 variant: regulation of its messenger level by xenobiotics

A rat liver cDNA library was prepared from total polyribosomal poly(A)+ RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or beta-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by beta-naphthoflavone treatment.