Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.     

Escherichia coli cells with mutations in the gene for adenylate cyclase (cya) exhibit a heat shock response

Abstract Adenylate cyclase mutants of Escherichia coli showed the heat-shock response. The heat-shock response was studied in two different mutants and in different growth media, including rich and minimal media. These results are in disagreement with the proposal that the cya gene regulates the expression of the heat-shock genes.     

Characterization of the lipopolysaccharides from Rhizobium meliloti strain 102F51 and its nonnodulating mutant WL113

Lipopolysaccharides from the Rhizobium meliloti wild-type strain 102F51, which is effective in symbiosis with alfalfa, and from the nonnodulating mutant WL113, defective in root hair adhesion, derived thereof, were isolated and comparatively analyzed. Both preparations were composed of galactose, glucose, glucuronic acid, galacturonic acid, glucosamine, 3-deoxyheptulosaric acid, and 2-keto-3-deoxyoctonic acid as the major sugar constitutents. After a modified methylation analysis (consisting of the following consecutive steps: methylation, carboxyl reduction, remethylation, mild acid hydrolysis, reduction, and trideuterio-methylation), all of the 3-deoxyheptulosaric and some of the 2-keto-3-deoxyoctonic acid residues were converted into their corresponding 3-deoxyalditol derivatives, which carried trideuteriomethyl groups at positions C-2, C-4, and C-6. Another part of the permethylated 3-deoxyoctitol was also found as 2,5,6- and 2,6,8-tri-O-trideuteriomethyl derivatives. NMR data obtained with the separated oligosaccharides and the results of methylation analysis indicated that the majority of 2-keto-3-deoxyoctonate was present in the fraction of permethylated disaccharide alditols, namely as 6-O-CD₃-aGlc(1→5)3-deoxyoctitol, 6-O-CD₃-βGlcNMeAcyl(1→4)3-deoxyoctitol, and as the permethylated trisaccharide alditol, αGalA(1→3)-[6-O-CD₃]-β-Glc(1→5)-[4-O-CD₃]-3-deoxyoctitol. The presence of trideuteriomethyl groups at C-4 of both 3-deoxyalditols and at C-6 of the glucosaminyl or glucosyl residues indicated the linkage points of the released acid-labile ketosidic substituents, such as 3-deoxyheptulosarate and 2-keto-3-deoxyoctonate, in these oligosaccharides. The main differences between the preparations from the wild-type 102F51 and its mutant strain WL 113 were found in the higher content (in strain 102F51) of the following oligosaccharides: α-glucuronosyl(1→4)2-keto-3-deoxyoctonate and α-galacturonosyl-(1→3)α-glucosyl-(1→5)2-keto-3-deoxyoctonate and in the decreased content of β-glucosaminyl(1→4)2-keto-3-deoxy-octonate. Received: 21 July 1995 / Accepted: 25 October 1995     

猪圆环病毒2型福建株的全基因组克隆与序列分析

根据GenBank上公布的猪圆环病毒2型全基因序列设计一对扩增PCV-2全基因的引物,建立扩增PCV-2全序列的PCR方法,并应用此方法从福建省不同地区采集的疑似断奶仔猪多系统衰竭综合征(PMWS)的仔猪肺组织病料中扩增出PCV-2全基因组(1 767 bp),将此基因片段克隆入pMD 18-T载体,筛选获得重组质粒pMD-PCV-2,并对其进行序列测定,然后对全基因组进行同源性和遗传进化分析。结果表明,福建省不同地区采集的猪肺组织扩增出的PCV-2全序列与GenBank上公布的的全基因组同源性介于94.9%-99.8%之间,ORF1的同源性介于97.2%-99.9%,ORF2的同源性也很高,介于97.7%-99.8%之间。其中福州株、福清株和漳州株与中国农大报道的12株、郑州株5株、杭州4株、武汉株3株、上海株2株、扬州2株、南京1株和兰州1株共30株在一个进化分支上,同源性也高达99.3%。本研究有助于监测PCV-2的疫源和进化关系,为进一步深入研究福建省生猪猪圆环病毒来源奠定一定基础。     

五步蛇蛇毒金属蛋白酶cDNA的克隆和序列分析

抽提五步蛇毒腺总RNA,通过反转录PCR(RT-PCR)扩增出五步蛇毒腺中一种低分子量金属蛋白酶(aculysinl)的cDNA,克隆到pGMT-vector并测定了全序列.推导其编码的蛋白质序列,发现aculysinl是以酶原形式合成的分泌蛋白,酶原包括信号肽、前肽、金属蛋白酶成熟肽和间隔肽4个部分.金属蛋白酶成熟肽与其它蛇毒金属蛋白酶相比,蛋白质一级结构具有一定的同源性,有一个保守的Zn2+结合位点:HEXXHXXGXXH.Aculysinl含有6个半胱氨酸,推测形成3对链内二硫键.五步蛇低分子量金属蛋白酶cDNA的克隆,为研究蛇毒金属蛋白酶结构与功能的关系,以及开发治疗血栓药物打下了良好的基础     

甜菜NADH-NR基因的克隆和序列分析

利用同源序列克隆方法从标准偏高糖型甜菜品种甜研7号(Ty7)中获得氯素诱导NADH-NR基因片段,通过RACE技术克隆NADH-NR基因全长序列.该基因ORF长度2 718 bp,编码905个氨基酸,包括147 bp的5'UTR和382 bp的3' UTR,GenBank上的注册号为EU163265,基因编码蛋白的等电点为6.12,推测分子量大小为102 kD,C端(778-891 aa)有一个跨膜区域.基因编码多肽含有3个氧化还原功能区:钼辅因子功能区(eukary NR Moco,93 - 478aa),Fe-血红素结合区(Cytb5,535 -608 aa),FAD结合区(FAD binding 6,653 -760 aa).在甜研7号NR下游的氨基酸残基中含有NADH-NR特有的CGPPP-M基序,说明该蛋白以NADH为电子供体.通过比对,甜研7号的NADH-NR基因与菠菜NADH-NR基因同源性最高,为86.18％.经Southem杂变检验,甜研7号中NADH-NR基因以低拷贝数存在.经基因组克隆分析,甜研7号NADH-NR含有3个内含子,4个外显子.     

Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba)

Insertion sequence (IS) hybridization was used to define the structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown under controlled conditions and inoculated with a suspension of the same soil. The detection of R. meliloti isolated from soil on agar plates was facilitated by use of a highly species specific DNA probe derived from ISRm5. All R. meliloti obtained directly from soil proved to be symbiotic (i.e. nodulated and fixed nitrogen with alfalfa). Analysis of 293 R. meliloti isolates revealed a total of 17 distinct IS genotypes of which 9, 9 and 15 were from soil, M. alba and M. sativa, respectively; 8 genotypes were common to soil and both plant species. The frequency of R. meliloti genotypes from soil differed markedly from that sampled from nodules of both legume species: 5 genotypes represented about 90% of the isolates from soil whereas a single genotype predominated among isolates from nodules accounting for more than 55% of the total. The distribution of genotypes differed between M. sativa and M. alba indicating species variation in nodulation preferences for indigenous R. meliloti. The data are discussed in the context of competition for nodulation of the host plant and the selection of Rhizobium strains for use in legume inoculants. This study has ecological implications and suggests that the composition of R. meliloti populations sampled by the traditionally used host legume may not be representative of that actually present in soil.     

Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase

The nucleotide sequence of the Bacillus anthracis edema factor (EF) gene (cya), which encodes a calmodulin-dependent adenylate cyclase, has been determined. EF is part of the tripartite protein exotoxin of B. anthracis. An ATG start codon, immediately upstream from codons which specify the first 15 amino acids (aa) of EF, was preceded by an AAAGGAGGT sequence which is its probable ribosome-binding site. Starting at this ATG codon, there was a continuous 2400-bp open reading frame which encodes the 800-aa EF-precursor protein with a Mr of 92,464. The mature, secreted protein (767 aa; Mr 88,808) was preceded by a 33-aa signal peptide which has characteristics in common with leader peptides for other secreted proteins of the Bacillus species. A consensus amino acid sequence (Gly-X-X-X-X-Gly-Lys-Ser,X = any aa), which was part of the presumed ATP binding site for EF, was also present. The codon usage of the EF gene reflected the high A + T (71%) base composition for its DNA. B. anthracis EF was not related to the Escherichia coli or yeast adenylate cyclases, but was related to the Bordetella pertussis calmodulin-dependent adenylate cyclase.     

A biological containment system for Rhizobium meliloti based on the use of recombination-deficient (recA−) strains

Abstract Using a newly developed integration vector, the Escherichia coli gusA gene conferring GUS-activity or the firefly ( Photinus pyralis ) luc gene mediating bioluminescence were integrated into a non-essential site of the chromosome of Rhizobium meliloti 2011. The integration of the constitutively expressed marker genes into the chosen site per se did not affect the strains' ability to perform homologous recombination, its growth characteristics or its symbiotic nitrogen fixation. Comparative microcosm analyses between the bioluminescent, recombination-proficient (RecA⁺) R. meliloti strain L33 whose construction is reported in this paper, and its previously described recombination-deficient (RecA⁻) isogenic counterpart L1 indicate that RecA⁻ strains of Rhizobium are safe hosts for deliberate release experiments.     

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1/ISRm7 and ISRm220-13-5 belong to a new family of insertion sequence elements

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1 and ISRm220-13-5 are 1481 and 1550 base pairs (bp) in size, respectively. ISRm102F34-1 is bordered by 15 bp imperfect terminal inverted repeat sequences (two mismatches), whereas the terminal inverted repeat of ISRm220-13-5 has a length of 16 bp (two mismatches). Both insertion sequence elements generate a 6-bp target duplication upon transposition. The putative transposase enzymes of ISRm102F34-1 and ISRm220-13-5 consist of 449 or 448 amino acid residues with predicted molecular weights of 50.7 or 51.3 kDa and theoretical isoelectric points of 10.8 or 11.1, respectively. ISRm102F34-1 is identical in 98.9% of its nucleotide sequence to an apparently inactive copy of an insertion sequence element, designated ISRm7, which flanks the left-end of the nodule formation efficiency (nfe) region of plasmid pRmeGR4b of S. meliloti strain GR4. ISRm102F34-1 and ISRm220-13-5 are closely related since they show an overall identity of 57.0% at the nucleotide sequence level and of 47.3% at the deduced amino acid level of their putative transposases. Both insertion sequence elements displayed significant similarity to the Xanthomonas campestris ISXc6 and its homolog IS1478a. Since none of these insertion sequence elements could be allocated to existing families of insertion sequence elements, a new family is proposed. Analysis of the distribution of ISRm102F34-1/ISRm7 in various local S. meliloti populations sampled from Medicago sativa, Medicago sphaerocarpa and Melilotus alba host plants at different locations in Spain revealed its presence in 35% of the isolates with a copy number ranging from 1 to 5. Furthermore, ISRm102F34-1/ISRm7 homologs were identified in other rhizobial species.     

The persistence of bioluminescent Rhizobium meliloti strains L1 (RecA-) and L33 (RecA+) in non-sterile microcosms depends on the soil type,on the co-cultivation of the host legume alfalfa and on the presence of an indigenous R. meliloti population

In comparative analyses the influence of soil type, the rhizosphere of plants and the presence of an indigenous R. meliloti population on the population dynamics of bioluminescent R. meliloti strains L1 (RecA-) and L33 (RecA+) was assessed in microcosm studies. Both strains established better in a loamy and a clayey soil compared to a sandy soil. RecA- strain L1 showed a slightly but statistically significant reduced survival ability compared to RecA+ strain L33 (p 0.05). The presence of the host plant alfalfa stimulated the growth of both strains in non-sterile soil and no differences in the survival between both strains were observed. Co-cultivation of clover or wheat plants, respectively, neither positively nor negatively influenced the strains' survival. The most pronounced effect on the survival of both strains was exerted by the presence of an indigenous R. meliloti population. RecA- strain L1 showed a significantly impaired survival compared to RecA+ strain L33 (p 0.002). Moreover, no growth stimulation of strains L1 and L33 by the presence of the host plant alfalfa could be observed. These results indicate that the recA mutation affects the long-term rather than the short-term persistence of R. meliloti after environmental release.     

Alamethicin or detergent permeabilization of the cell membrane as a tool for adenylate cyclase determination: Application to the study of hormone responsiveness in lymphocytes

Treatment of mouse lymphocytes with very low concentrations of alamethicin or Lubrol PX induces spontaneous permeabilization of the plasma membrane to ATP and allows determination of adenylate cyclase activity in whole cells. The permeabilized cells retain responsiveness to hormones (isoproterenol, adenosine analogs) and to fluoride. The main advantage of this new method is that it does not require any homogenization step, and thus adenylate cyclase activities can be accurately and reproducibly measured with very low amounts of cells. It should be especially useful for the study of purified lymphocyte subpopulations.     

苦荞谷胱甘肽转移酶(FtGST)基因的克隆与序列分析

采用RACE技术,从苦荞(Fagopyrum tatarium)中克隆得到一个谷胱甘肽转移酶(Glutathione S-transferase protein,FtGST)基因。序列分析表明,FtGST基因全长DNA序列和cDNA序列编码区分别为746 bp和666 bp,DNA序列含有一个长度为80 bp(342-421 bp)的内含子;开放阅读框(ORF)长666 bp,编码221个氨基酸。生物信息学分析表明,FtGST基因推导的蛋白质含有Tau家族典型的底物结合口袋、谷胱甘肽结合位点(G-site)和疏水性底物结合位点(H-site)氨基酸残基,表明FtGST为Tau家族蛋白。     

牦牛SRY和TRO基因部分序列克隆与测序分析

对牦牛SRY和TRO的部分基因克隆和序列分析,以期为进一步开展该基因与其性别相关分析,进行性染色体的基因定位、以及分子标记辅助选择等研究提供了理论依据。用特定引物对牦牛和西门塔尔牛的SRY、TRO基因部分序列进行扩增并进行TA克隆和测序。通过测序结果与普通牛的比对分析表明,这两个基因区域在牛种中有极高的保守性。牦牛与普通牛SRY和TRO基因这两个区域的核酸同源性分别达到了99．08％和99．39％。根据对这两个基因序列的研究为精子或者胚胎的性别鉴定提供有力的理论基础。     

Effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (synthetic platelet-activating factor)

We have investigated the effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (1-octadecyl-2-acetyl-G-3-PC), using particulate fractions from human and rabbit platelets that had been frozen and thawed in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetate to prevent Ca²⁺-dependent proteolysis. When 10 μM GTP was present, 100 mM NaCl stimulated the activity of the rabbit enzyme 5.6-fold and that of the human enzyme 2.2-fold. Under these conditions, maximum inhibitions of 90% and 64% were obtained on addition of 100 nM 1-octadecyl-2-acetyl-G-3-PC to rabbit and human preparations, respectively. These inhibitions resulted partly from an NaCl-independent inhibition of basal enzyme activity and partly from reversal of the stimulatory effect of NaCl. The relative abilities of the chlorides of different monovalent cations to enhance inhibition of rabbit platelet adenylate cyclase were: NaCl >LiCl >KCl >choline chloride. NaCl also increased the concentrations of 1-octadecyl-2-acetyl-G-3-PC required for half-maximal inhibition of adenylate cyclase but this action of NaCl did not correlate with its stimulatory effect on enzyme activity. After particulate fractions from platelets of either species were washed, 10 μM GTP inhibited basal adenylate cyclase activity in the absence of NaCl but stimulated the enzyme in the presence of NaCl. Inhibition of adenylate cyclase by 1-octadecyl-2-acetyl-G-3-PC was then either enhanced by GTP (rabbit material) or completely dependent on added GTP (human material). Stimulation of the activity of the washed human preparations by NaCl required GTP, but concentrations lower than required for potentiation of the inhibitory effect of 1-octadecyl-2-acetyl-G-3-PC by NaCl were effective.     

Characterization of the norepinephrine-activation of adenylate cyclase suggests a role in memory affirmation pathways: Overexposure to epinephrine inactivates adenylate cyclase,a causal pathway for stress-pathologies

Incubation with noradrenaline (norepinephrine) of isolated membranes of rat's brain corpus striatum and cortex, showed that ionic-magnesium (Mg²⁺) is required for the neurotransmitter activatory response of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1)], AC.     

Guanylyl cyclases in the tropical land crab, Gecarcinus lateralis: Cloning of soluble (NO-sensitive and -insensitive) and membrane receptor forms

cDNAs encoding three guanylyl cyclases (GCs), which catalyze the production cGMP from GTP, were cloned from the blackback land crab, Gecarcinus lateralis: the β subunit of a NO-sensitive soluble GC (Gl-GC-Iβ; 2380 bp; 603 amino acids; 68,435 Da), a membrane receptor GC (Gl-GC-II; 4609 bp; 1349 amino acids; 150,999 Da), and a NO-insensitive soluble GC (Gl-GC-III; 1416 bp; 285 amino acids; 32,487 Da). All three have a highly-conserved catalytic domain located in the C-terminal region and are therefore likely to be enzymatically active. Gl-GCIβ contains heme/NO-binding (HNOB) and HNOB-associated domains characteristic of the catalytic subunit of NO-sensitive GCs. Gl-GC-II encodes a single-pass membrane protein containing ligand-binding (LB), transmembrane (TM), kinase homology (KH), and dimerization domains. Gl-GC-III is similar to Gl-GC-II but lacks the LB, TM, and most of the KH domains. Isoforms of Gl-GC-Iβ and Gl-GC-II appear to be generated by alternative splicing. Two Gl-GC-Iβ isoforms differed in the absence (Δ32N) or presence (Δ0N) of a 32-amino acid sequence at the N-terminus. The truncated form may not bind a heme group, but still would be able to dimerize with the alpha subunit to form a NO-insensitive enzyme. Three Gl-GC-II isoforms varied in length within the N-terminal ligand-binding domain (+ 18, + 9, and + 0 amino acid insertions). As GC-II is the putative receptor for crustacean hyperglycemic hormone (CHH), these data suggest that the isoforms differ in binding to CHH and related neuropeptides.     

Effects of HIS1 modifications on the ability of vasoactive intestinal peptide to stimulate adenylate cyclase from rat and human tissues

The importance of the N-terminal His residue of VIP for stimulating adenylate cyclase was appreciated by estimating the intrinsic activity and EC50 of four VIP analogues on membranes from rat lung, liver, brain, anterior pituitary, and pancreas, and on human heart membranes. In all tissue preparations tested except one, the order of efficacy (and often potency) was: VIP greater than (Ac-His1)VIP greater than (Phe1)VIP = (3-Me-His1)VIP greater than (D-His1)VIP. In rat heart membranes, the order of efficacy was somewhat different: VIP greater than (Ac-His1)VIP = (Phe1)VIP greater than (D-His1)VIP greater than (3-Me-His1)VIP. These data demonstrated the key role of His1 in VIP in activating adenylate cyclase. They suggest that a given VIP analogue might act as full agonist in tightly coupled adenylate cyclase systems (such as those of rat lung and liver membranes) whereas the same analogue could not promote full activity in poorly coupled systems (such as that present in rat brain synaptic membranes).