水稻线粒体DNA雄性不育有关特异片段的克隆及序列分析

应用任意单引物聚合酶链反应技术,从水稻ＷＡ 型雄性不育系的线粒体ＤＮＡ 中得到一个特异的扩增片段Ｒ２－６３０ ＷＡ。以该片段为探针进行Ｓｏｕｔｈｅｒｎ 杂交分析检测到在雄性不育胞质与正常可育胞质间存在的线粒体ＤＮＡ 多态性。不育系珍汕９７Ａ 和其Ｆ１ 杂种的杂交图谱相同。而保持系珍汕９７Ｂ和恢复系明恢６３ 的杂交图谱一样。序列测定该片段全长６２９ ｂｐ,其碱基组成Ａ＋ Ｔ＝ ５４．１％ ,同源性比较结果显示, 该片段与１２３６ 个已报道的植物基因（包括１６ 个水稻线粒体基因）序列的同源性均小于５０％ 。序列内含有一个长度为１０ ｂｐ 的反向重复序列５－ＡＣＣＡＴＡＴＧＧＴ－３,位于２６２—２７２ 区段。另外,其３７９—４３９ 区段可编码一个含２０个氨基酸残基的短肽。上述结果表明,Ｒ２－６３０ ＷＡ 片段确与水稻野败型雄性不育密切相关。推测反向重复序列５－ＡＣＣＡＴＡＴＧＧＴ－３在细胞质雄性不育性状形成中,可能起着重要作用     

Generation of anNco I restriction site for translational fusions using PCR-mediated,site-directed mutagenesis

A polymerase chain reaction-based method of site-directed mutagenesis was used to introduce anNco I restriction site on the translation start site of a tomato peroxidase gene. This quick and efficient method utilized two overlapping synthetic oligonucleotide primers containing the requisite base pair changes on the ATG translation start site and two flanking primers in PCR. The resulting DNA amplified fragments were fused together byNco I digestion at the mutated ends followed by a T4 ligation reaction. A rapid alternative method utilizing the overlapping fragments and the flanking primers in PCR can also be used for ligating the two fragments. Cloning and sequencing of the PCR-amplified fragments provided additional evidence for the presence of the site-specific mutations. Unique restriction sites upstream and downstream of the site-specific mutation allows for the easy transfer of this mutated region into the wild type peroxidase gene.     

Mammalian expression of single chain variable region fragments dimerized by Fc regions

To promote application of a single chain variable region fragment (sFv) in immunoglobulins, a sFv gene was connected to an IgG1 Fc gene, designated as a sFvc gene, and used for transfection of Sp2/0. As a result, the sFvc protein was found to be secreted in a dimeric form. It is thus felt that the sFvc protein, which mimicks the shape of a naturally occurring antibody, can be simple and useful to reproduce divalency and Fc-associated effecter functions as seen in a natural antibody.Abbreviations Abbreviations sFv single chain variable region fragment - Fc constant region of immunoglobulin - sFvc single chain variable region fragment with an Fc region     

Effects of dispersal in a tri-trophic metapopulation model

The aim of this paper is to understand how dispersal in a patchy environment influences the stability properties of tri-trophic metapopulations. Differential equation models for tri-trophic metapopulations are formulated and analysed. The patchy nature of the metapopulations is incorporated through dispersal phases. Two variants are studied: one with a dispersal phase for the top and one with a dispersal phase for the middle level. A complete characterisation of stable and unstable equilibria is given and the possibility of invasion in these food chains is studied. A dispersal phase for the middle level can destabilize the bottom level-middle level interaction, because of the delay that dispersal causes in the reaction to the resource. When the middle level is not efficiently controlled by the top level, the unstable bottom level-middle level pair can destabilize the entire food chain. Dispersal for the top level can destabilize in the same way. A characterisation of the long term behaviour of the models is given. Bistability with a stable three species equilibrium and a stable limit cycle is one of the possibilities.     

Cold-sensitive conditional mutations in Era, an essential Escherichia coli GTPase, isolated by localized random polymerase chain reaction mutagenesis

Abstract Conditional cold-sensitive mutations in Era, an essential Escherichia coli GTPase, were isolated. Localized random polymerase chain reaction (PCR) mutagenesis employing Taq and T7 DNA polymerases under error prone amplification conditions was exploited to generate mutations in the era gene. A plasmid exchange technique was used to identify conditional cold-sensitive mutations in Era that give rise to defective cell growth below 30 °C. Three recessive missense mutations in Era, N26S, A156D, and E200K, were isolated. All three mutations are located at residues conserved in Era homologues from Streptococcus mutans and Coxiella burnetii .     

Control of glyphosate uptake and metabolism in Pseudomonas sp. 4ASW

Abstract The tandem mini-exon gene repeat is an ideal diagnostic target for trypanosomatids because it includes sequences that are conserved absolutely coupled with regions of extreme variability. We have exploited these features and the polymerase chain reaction to differentiate Phytomonas strains isolated from phloem, fruit or latex of various host plants. While the transcribed regions are nearly identical, the intergenic sequences are variable in size and content (130–332 base pairs). The mini-exon genes of these phytomonads can therefore be distinguished from each other and from the corresponding genes in insect trypanosomes, with which they are oft confused.     

Characterization of Superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers

Abstract An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae , and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes , and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.     

Transfer and expression of the human multiple drug resistance gene as potential human gene therapy

The human multiple drug resistance (MDR) gene has been used as a model for human gene transfer which could lead to human gene therapy. MDR is a transmembrane protein which pumps a number of toxic substances out of cells including several drugs used in cancer chemotherapy. Normal bone marrow cells express low levels of MDR and are particularly sensitive to the toxic effects of these drugs. There are two general applications of MDR gene therapy: (1) to provide drug-resistance to the marrow of cancer patients receiving chemotherapy, and (2) as a selectable marker which when co-transferred with a non-selectable gene such as the human beta globin gene can be used to enrich the marrow for cells containing both genes. We demonstrate efficient transfer and expression of the human MDR gene in a retroviral vector into live mice and human marrow cells including CD34⁺ cells isolated from marrow and containing the bulk of human hematopoietic progenitors. MDR gene transduction corrects the sensitivity of CD34⁺ cells to taxol, an MDR drug substrate, and enriches the marrow for MDR-transduced cells. The MDR gene-containing retroviral supernatant used has been shown to be safe and free of replication-competent retrovirus. Because of the safety of the MDR retroviral supernatant, and efficient gene transfer into mouse and human marrow cells, a phase 1 clinical protocol for MDR gene transfer into cancer patients has been approved to evaluate MDR gene transfer and expression in human marrow.     

Addition of side chain interactions to modified Lifson-Roig helix-coil theory: application to energetics of phenylalanine-methionine interactions.

We introduce here i, i + 3 and i, i + 4 side chain interactions into the modified Lifson-Roig helix-coil theory of Doig et al. (1994, Biochemistry 33:3396-3403). The helix/coil equilibrium is a function of initiation, propagation, capping, and side chain interaction parameters. If each of these parameters is known, the helix content of any isolated peptide can be predicted. The model considers every possible conformation of a peptide, is not limited to peptides with only a single helical segment, and has physically meaningful parameters. We apply the theory to measure the i, i + 4 interaction energies between Phe and Met side chains. Peptides with these residues spaced i, i + 4 are significantly more helical than controls where they are spaced i, i + 5. Application of the model yields delta G for the Phe-Met orientation to be -0.75 kcal.mol-1, whereas that for the Met-Phe orientation is -0.54 kcal.mol-1. These orientational preferences can be explained, in part, by rotamer preferences for the interacting side chains. We place Phe-Met i, i + 4 at the N-terminus, the C-terminus, and in the center of the host peptide. The model quantitatively predicts the observed helix contents using a single parameter for the side chain-side chain interaction energy. This result indicates that the model works well even when the interaction is at different locations in the helix.