A general method for site-directed mutagenesis in prokaryotes

The genetic analysis of genes from prokaryotic species for which experimental genetic systems have not yet been developed is often limited by the difficulty of producing mutations in those genes. We report here a general technique applicable to Gram-negative prokaryotes for site-directed mutagenesis of cloned DNA fragments which we have applied to the study of the symbiotic nitrogen fixation genes of Rhizobium meliloti. In particular, we mutagenized cloned R. meliloti restriction fragments in Escherichia coli with transposon Tn5 and then replaced the wild-type parental DNA sequences with the mutant DNA sequences in the R. meliloti genome. Using this method we show that an R. meliloti DNA restriction fragment, cloned previously on the basis of homology to Klebsiella pneumoniae nif genes, contains gene(s) essential for symbiotic nitrogen fixation. In addition, we use this method to construct a physical genetic map of a subset of the R. meliloti nif genes.     

利用反义RNA探索外源基因丢失的分子机理

利用反义ＲＮＡ技术研究了调控ＰＡＲＰ酶基因的表达对外源基因整合稳定性的影响。将ＰＡＲＰ基因ｃＤＮＡ的部分序列反向插入到真核表达载体ｐＳＭＧ中,将重组质粒分别导入携带有外源基因的细胞中,地塞米松诱导反义ＰＡＲＰ基因的表达后,进行Ｓｏｕｔｈｅｒｎ杂交检测。结果表明,外源基因仍保留在基因组中,这意味着外源基因的丢失并不是由于单一ＰＡＲＰ酶活性降低所致。     

Present status and development on biological nitrogen fixation research in China

This presentation introduces the advances in biological nitrogen fixation research abroad, in particular,describes the great progress and achievements on its research in China as follows: collection of rhizobial resources and establishment of the largest database of Rhizobium in China, correction and development of Rhizobium taxonomy in international; discovery of a couple of nif genes, identification and unification of linkage among the nif gene operons of Klebsiella pneumoniae, finding of regulative mechanism of positive regulation nif gene and its sensitivity to oxygen,temperature; finding of the activity of nodulation gene nodD3 product in Sinorhizobium meliloti which is not controlled by flavonoid produced from its host alfalfa; finding of the association between expression of genes coding the products for carbon utilization and nitrogen metabolism and their regulations; chemical synthesis of nodulation factor of Sinorhizobium meliloti; constructions of engineered nitrogen fixers and utilization in practice based on the research of gene expression and regulation; chemical simulation of the structure and function of nitrogenase and bringing forward the model of nitrogenase active center for the first time in international and synthesis of model compounds which were paid attention by colleagues abroad. Finally, the development of nitrogen fixation research in China in future has been put forward, suggesting that the nif gene regulation and its role in providing crops with nitrogen element, signal transduction and molecular interactions between Rhizobium and legume, coupling between carbon and nitrogen metabolisms, nitrogen fixation and photosynthesis, and functional genomics of nitrogen-fixing nodule symbiosis, etc., would be actively worked on.     

Transcriptome analysis of Sinorhizobium meliloti nodule bacteria in nifA mutant background

Sinorhizobium meliloti is one genus of gram-nega- tive soil bacteria that can fix atmospheric nitrogen in root nodules of its symbiotic leguminous host plants[1]. Specific recognition and progressive differentiation ofboth bacteria and host cells are requ…     

Tissue-specific expression of rat myosin light-chain 2 gene in transgenic mice

One approach to determining how the differential expression of specific genes is regulated in higher organisms is to introduce cloned copies of the genes (or parts of the genes) into the genomes of individual organisms from the very beginning of their development. The way in which the exogenous genetic information behaves during the development of the experimental organisms can then provide a means of defining the DNA sequences that restrict the expression of the gene to specific cell types and times of development. So far, several different genes have been introduced into the genomes of mice, but in only a few cases have the exogenous genes retained the tissue specificity of expression of the equivalent endogenous genes. I report here that in two out of three 'transgenic' mice carrying copies of the rat gene for skeletal muscle myosin light chain 2, the exogenous gene is expressed specifically in skeletal muscle cells. The sequences contained in the cloned copy of the myosin light-chain 2 gene used in these experiments are thus sufficient to confer a tissue-specific pattern of expression.     

Progress in artificial control system for gene expression

Along with the increasingly wide application of transgenic techniques, new stricter criteria have been raised for controlling the expression of exogenous genes. For these demands, a series of artificial control systems for gene expression have been developed and testified in recent years, which can control exogenous genes expression in exact time and certain level by administration of a specific drug or hormone. The successful construction of these systems offers a practicable method to control precise expression of exogenous gene in organisms, and raises the feasibility of wide application of gene therapy.     

Sinorhizobium meliloti nifA gene exerts a pleiotropic effect on nodulation through the enhanced plant defense response

Sinorhizobium meliloti nifA gene is required for the expression of a bunch of nif and fix genes. Here, we report its pleiotropic effects on the nodule formation. Compared with wild type strain, nifA mutant sig- nificantly reduced nodule suppression rate in split-root system. The plants inoculated with mutant strain produced lower amount of daidzein and less necrotic cells on their roots. In addition, the defense genes failed to be evoked by nifA mutant at the early nodulation stage. These findings indicated that host defense response was one of the mechanisms mediated by nifA gene to regulate nodule formation during symbiosis. Even though nifA mutant could increase the number of nodules in host plant, it synthesized lower Nod factors than wild type. This suggested that nifA gene mediated multiple and diverse instances in nodulation formation.     

MtROP8 is involved in root hair development and the establishment of symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti

Emerging evidence has demonstrated that ROP GTPases play important roles in symbiosis, but the molecular mechanisms on their regulation in symbiosis are largely unknown. In this study, we showed that MtROP8 is involved in the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti. Expression analyses showed that MtROP8 was down-regulated in the early infected roots, but significantly up-regulated in nodules compared to the roots. Phenotypic analysis of RNA inter- ference （RNAi）-mediated silencing of MtROP8 revealed that knock-down of MtROP8 expression resulted in various developmental defects of root hairs, including branched hairs, short bulbous root hairs, and even root hairs with apparent swollen bases, which were caused by the modi- fication of the distribution and the levels of reactive oxygen species （ROS）. Moreover, infection events were increased in transgenic roots harboring the MtROP8 RNAi construct in response to S. meliloti inoculation, concomitant with enhanced nodulation. These results indicate that MtROP8 participates in root hair development and the establishmentof the symbiotic interaction by regulating ROS production and distribution.     

A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti.

The expression of the nitrogen-fixation genes of Rhizobium meliloti is controlled by oxygen. These genes are induced when the free oxygen concentration is reduced to microaerobic levels. Two regulator proteins, FixL and FixJ, initiate the oxygen-response cascade, and the genes that encode them have been cloned. The fixL product seems to be a transmembrane sensor that modulates the activity of the fixJ product, a cytoplasmic regulator. FixL and FixJ are homologous to a family of bacterial two-component regulators, for which the mode of signal transduction is phosphorylation. We report here the purification of both FixJ and a soluble truncated FixL (FixL*), overproduced from a single plasmid construct. FixL* catalyses its own phosphorylation and the transfer of the gamma-phosphate of ATP to Fix J. The resulting FixJ-phosphate linkage is sensitive to base, as are the aspartyl phosphates of homologous systems. Visible spectra of purified FixL* show that it is an oxygen-binding haemoprotein. We propose that FixL senses oxygen through its haem moiety and transduces this signal by controlling the phosphorylation of FixJ.     

一种新型叶绿体表达系统的构建

针对目前的叶绿体转基因技术存在载体构建步骤繁琐耗时的问题,建立了一种新型的不需自带启动子的叶绿体表达系统,选择模式植物烟草叶绿体中的假基因作为外源基因的插入位点.通过基因枪转化法获得4株独立的叶绿体转化植株.此新技术可实现叶绿体载体构建的时间、技术成本最小化,从而促进转基因叶绿体的应用.     

Sinorhizobium meliloti lsrB is involved in alfalfa root nodule development and nitrogen-fixing bacteroid differentiation

Rhizobia interact with host legumes to induce the formation of nitrogen-fixing nodules, which is very important in agriculture and ecology. The development of nitrogen-fixing nodules is stringently regulated by host plants and rhizobial symbionts. In our previous work, a new Sinorhizobium meliloti LysR regulator gene (lsrB) was identified to be essential for alfalfa nodulation. However, how this gene is involved in alfalfa nodulation was not yet understood. Here, we found that this gene was associated with prevention of premature nodule senescence and abortive bacteroid formation. Heterogeneous deficient alfalfa root nodules were induced by the in-frame deletion mutant of lsrB (lsrB1-2), which was similar to the plasmid-insertion mutant, lsrB1. Irregular senescence zones earlier appeared in these nodules where bacteroid differentiation was blocked at different stages from microscopy observations. Interestingly, oxidative bursts were observed in these nodules by DAB staining. The decreased expression of lipopolysaccharide core genes (lpsCDE) was correspondingly determined in these nodules. S. meliloti lipopolysaccharide is required for suppression of oxidative bursts or host cell defense. These findings demonstrate that the S. meliloti lsrB gene is involved in alfalfa root nodule development and bacteroid differentiation by suppressing oxidative bursts or defense responses in host cells.     

A haemoglobin switching activity modulates hereditary persistence of fetal haemoglobin

During human development there is a switch from fetal to adult haemoglobin formation, reflecting the differential expression of fetal (G gamma and A gamma) and adult (beta and delta) globin genes. Mutations that inhibit this switch produce variants of the syndrome of hereditary persistence of fetal haemoglobin (HPFH). Adult heterozygotes for these mutants produce 15-30% fetal haemoglobin (HbF) in their red cells. The general assumption is that the mutations result in a permanent switching on of gamma-globin genes. Here, however, we show that fetal globin expression can be turned off in cultures of HPFH cells by an uncharacterized factor in fetal sheep serum. This is the first demonstration that mutations affecting the developmental expression of globin genes can be modulated by exogenous factors. The findings raise the possibility that the phenotype of HPFH is not simply the direct result of mutations in or around globin genes but the consequence of the mutations on the interaction of globin genes with trans-acting regulatory factors.