Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata

Biological nitrogen fixation requires the nitrogenase enzyme complex, ATP, and a strong reductant. Klebsiella pneumoniae contains 15 linked nitrogen fixation (nif) genes, three of which, nifH, nifD and nifK have been sufficiently conserved in evolution that cloned K. pneumoniae nifHDK DNA will hybridize to DNA sequences from every nitrogen-fixing bacterium examined to date, including the purple, non-sulphur bacterium Rhodopseudomonas capsulata, in which one complete nifHDK operon has been mapped. Using cloned K. pneumoniae nifHDK DNA we report here that R. capsulata contains multiple copies of the genes for nitrogenase components. Two regions containing sequences homologous to all three nif structural genes have been identified, and mutations in one region produced a Nif- phenotype. Nif+ pseudorevertants were derived from these mutants, some of which retained the original mutation suggesting that some of the extra nif gene sequences can be functionally activated.     

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.     

豆科植物-根瘤菌共生固氮的分子机理

与豆科植物－根瘤菌共生固氮有关的基因涉及根瘤菌基因和宿主基因，根瘤菌基因有结瘤基因（nodD,nodAB-CIJ和hsn基因），根瘤菌细胞表面结构基因（exs,lps和ndv基因）和固氮基因（nif和fix基因）；宿主基因主要是结瘤素基因（ENOD和NOD基因）。根瘤菌结瘤基因表达后诱导产生结瘤因子。在根瘤发育过程中，这些基因在根瘤菌与植物之间进行着信息交换，并且具有不同的表达水平。结瘤因子和植物激素对它们进行着调节。     

Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal

Rhizobia are symbiotic bacteria that elicit the formation on leguminous plants of specialized organs, root nodules, in which they fix nitrogen. In various Rhizobium species, such as R. leguminosarum and R. meliloti, common and host-specific nodulation (nod) genes have been identified which determine infection and nodulation of specific hosts. Common nodABC genes as well as host-specific nodH and nodQ genes were shown recently, using bioassays, to be involved in the production of extracellular Nod signals. Using R. meliloti strains overproducing symbiotic Nod factors, we have purified the major alfalfa-specific signal, NodRm-1, by gel permeation, ion exchange and C18 reverse-phase high performance liquid chromatography. From mass spectrometry, nuclear magnetic resonance, (35)S-labelling and chemical modification studies, NodRm-1 was shown to be a sulphated beta-1,4-tetrasaccharide of D-glucosamine (Mr 1,102) in which three amino groups were acetylated and one was acylated with a C16 bis-unsaturated fatty acid. This purified Nod signal specifically elicited root hair deformation on the homologous host when added in nanomolar concentration.     

onc sequences (v-fes) of Snyder-Theilen feline sarcoma virus are derived from noncontiguous regions of a cat cellular gene (c-fes)

Type C sarcoma viruses are genetic recombinants containing portions of replication-competent helper viruses linked to sarcoma virus-specific sequences (generically designated onc genes) which are thought to be required for acute fibroblast transformation. The onc elements of different avian and mammalian sarcoma viral isolates are each homologous to subsets of cellular DNA sequences which have no well-defined role in normal cells. Because of the lack of significant homology between helper viral genes and cellular onc sequences, the recombinational mechanisms which facilitate the formation of sarcoma viral genomes remain unclear. In Moloney murine sarcoma virus, viral onc (or v-mos) and cellular onc (or c-mos) sequences exhibit complete and uninterrupted homology as determined by heteroduplex and restriction enzyme analyses of molecularly cloned DNA. By contrast, the cellular counterparts of the onc elements of Rous sarcoma virus (G. Cooper and R. Parker, personal communication), avian erythroblastosis virus (B. Vennstrom, personal communication), Abelson leukaemia virus (D. Baltimore, personal communication), Harvey sarcoma virus (E. Scolnick, personal communication) and simian sarcoma virus (R. Gallo, personal communication) are now known to contain intervening sequences which do not appear in the respective viral genomes. Here we report the use of the Southern blot technique to examine cat cellular DNA sequences (c-fes) homologous to the onc gene (v-fes) of Snyder-Theilen feline sarcoma virus (ST-FeSV). We used cloned DNA 'probes' containing defined portions of the ST-FeSV genome to show that v-fes sequences originate from at least four noncontiguous sequences in cat cellular DNA, separated from each other by intervening sequences.     

Different recombination site specificity of two developmentally regulated genome rearrangements

In the absence of a combined nitrogen source, such as ammonia, approximately every tenth vegetative cell along filaments of the cyanobacterium Anabaena develops into a heterocyst, a terminally differentiated cell that is morphologically and biochemically specialized for nitrogen fixation. At least two specific DNA rearrangements involving the nitrogen-fixation (nif) genes occur during heterocyst differentiation, one within the nifD gene and the other near the nifS gene. The two rearrangements have several properties in common. Both occur quantitatively in all heterocyst genomes, both occur at approximately the same developmental time, late in the process of heterocyst differentiation, and both result from site-specific recombination between short repeated DNA sequences. We report here the nucleotide sequences found at the site of recombination near the nifS gene. These sequences differ from those found previously for the nifD rearrangement, suggesting that the two rearrangements are catalysed by different enzymes and may be regulated independently. We also show that the nifS gene is transcribed only from rearranged genomes.     

Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena

Nitrogen fixation by the cyanobacterium Anabaena is carried out in heterocysts, specialized, non-dividing cells which differentiate under conditions of ammonia or nitrate deprivation. In Anabaena, heterocyst differentiation is accompanied by rearrangement of some nitrogen fixation genes. A site-specific recombination between an 11 base-pair direct repeat sequence flanking the nifK and nifD genes removes 11 kilobases of intervening DNA, resulting in juxtaposition of the two genes and an alteration of the nifD protein-coding sequence.     

ATP sulphurylase activity of the nodP and nodQ gene products of Rhizobium meliloti

The symbiotic bacterium Rhizobium meliloti stimulates alfalfa (Medicago sativa L.) roots to undergo morphogenesis and form nitrogen-fixing nodules. It has been proposed that the bacterial genes nodABC, common to all Rhizobium, are required for synthesis of an oligosaccharide factor, which is converted to a sulphated form (NodRm-1) by the products of the R. meliloti-specific genes nodH and nodQ1-5; NodRm-1 elicits host-specific plant responses. Previously we have shown that the nodP gene is homologous to a segment of the Escherichia coli genome; when we cloned this E. coli fragment we found that it mapped near 59 minutes, corresponding to the cysDNC locus. The genes cysD and cysN encode proteins that catalyse the synthesis of adenosine 5'-phosphosulphate, the first step in the activation of inorganic sulphate. Here we demonstrate that nodP and nodQ correspond to cysD and cysN, and that their proteins have ATP sulphurylase activity both in vivo and in vitro. We propose that nodP and nodQ synthesize an activated sulphate that is an intermediate in the formation of the alfalfa-specific sulphated nodRm-1 factor.     

Regulation of nitrogen metabolism genes by nifA gene product in Klebsiella pneumoniae

The Klebsiella pneumoniae nifA gene product, which is known to activate expression of the nitrogen fixation (nif) structural genes, is shown here also to be able to substitute for the product of the gene glnG (ntrC) in the regulation of other nitrogen metabolism genes. An evolutionary relationship between the nifA and glnG genes is suggested.     

Structure of a family of rat amylase genes

The sequences of two cloned rat pancreatic amylase cDNAs comprising 95% of the mRNA sequence are reported. Analysis of cloned rat genomic DNA fragments using cloned cDNA probes indicates that the rat genome contains multiple closely related amylase genes in which the cDNA sequences are distributed within a region 9 kilobases in length and are interrupted by at least seven intervening sequences.     

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…     

海甘蓝硫甙合成关键酶S—GT基因克隆及种子特异性hpRNAi载体构建

根据甘蓝型油菜S-GT（thiohydroximate S-glucosyltransferase）基因cDNA序列设计引物,以海甘蓝总DNA为模板进行PCR扩增,获得S-GT基因全长。克隆的海甘蓝S—GT序列与甘蓝型油菜序列相比,除74bp的内含予部分外有92个碱基的差别,相似性高达93．4％。分析显示该序列均有完整的开放阅读框,并表明所克隆的海甘蓝S-GT序列编码465个氨基酸,在第10个位点上比甘蓝型油菜序列少一个丙氨酸（A）,总共有23个氨基酸不同,相似性为95．06％。根据获得的基因序列设计引物扩增出同一基因序列相同但是带有不同酶切位点的两个片段,将两个片段反向插入到已构建的带有种子特异表达载体内含子的两端,成功构建了海甘蓝S-GT基因的种子特异性hpRNAi载体,为特异性降低海甘蓝的种子硫甙奠定了基础。     

硅胶破碎法抽提真菌染色体DNA

在进行基因组步行PCR克隆真菌木聚糖酶基因的研究中,探索并建立了一套可在普通分子生物学实验室采用的高得率、高质量真菌染色体DNA的抽提方法.采用液氮研磨和硅胶破碎相结合提高染色体DNA得率,采用亚精胺法纯化提高DNA的纯度.抽提的染色体DNA用琼脂糖凝胶电泳、限制酶切、PCR反应及紫外吸收光谱等方法进行了鉴定,结果显示此方法可以获得分子量大、样品纯的染色体DNA,可有效地用于PCR扩增,并能被限制酶有效地消化.     

Investigation of genetic linkage between myosin and actin genes using an interspecific mouse back-cross

The introduction of cloned probes to follow the segregation of DNA restriction fragment length polymorphisms (RFLPs) has led to a revival of mendelian genetics in attempts to map the human genome. In the mouse, however, it has often proved difficult to detect an RFLP with a DNA probe between different inbred strains of the laboratory mouse. To circumvent this problem, we have used two species, Mus musculus domesticus and Mus spretus which interact as sympatric species but can be interbred under laboratory conditions. Because of the relative evolutionary distance between these species, they exhibit polymorphism at many more loci than do different strains of the usual M. m. domesticus laboratory mouse. This is also observed at the DNA level when the sizes of restriction fragments encoding a specific gene are compared. We have used these RFLPs between M. m. domesticus and M. spretus to follow the segregation of genes encoding different isoforms of myosin alkali light chains in the backcross progeny between these species and to compare this with that of other contractile protein genes. No linkage between these genes was observed.     

Comparative chromosome mapping of a conserved homoeo box region in mouse and human

Specific genes are assumed to regulate pattern formation in the mammalian embryo, but as yet none has been identified unequivocally. It is possible that such genes in mammals may be identified by virtue of a conserved coding sequence, because many of the Drosophila melanogaster homoeotic and segmentation genes, which have crucial roles in the regulation of segmental pattern formation during embryonic development, contain a 180-base pair (bp) DNA sequence, the homoeo box, and that sequences homologous to the Drosophila homoeo box are also present in 6-10 copies in higher animals, including mammals. Although the assumption that the homoeo box identifies genes responsible for pattern formation in mammals remains to be validated, it is a particularly attractive hypothesis given the strong conservation of homoeo boxes over vast evolutionary distances. Here we report the localization of a human homoeo box region, previously cloned and shown to contain two homoeo boxes within a sequence of 5-kilobases (kb), to the long arm of chromosome 17. We show that two single-copy homoeo box-flanking probes derived from this region strongly hybridize to single-copy restriction fragments in mouse genomic DNA and that these conserved homoeo box-flanking sequences map to mouse chromosome 11. This may be significant as several genes that map to chromosome 17 in human also map to chromosome 11 in the mouse, implying that a segment of mouse chromosome 11 is homologous to a region of human chromosome 17. Taken together, these data suggest that the homoeo box region detected with our probes is highly conserved in human and mouse.