Unusually large telomeric repeats in the yeast Candida albicans

We have identified sequences at the telomeres of the yeast Candida albicans and have found that they are composed of tandem copies of a 23-bp sequence. Through the cloning of native telomeric ends and the characterization and cloning of a "healed" end, we demonstrate that these repeated sequences are sufficient to function as a telomere. All copies of the 23-bp repeat that have been sequenced from a number of C. albicans strains are identical. In contrast, adjacent subtelomeric sequences are variable both between strains and within the WO-1 strain. In the WO-1 strain, the lengths of the telomeres are dependent upon growth temperature and are substantially longer at higher temperatures. Telomere growth is accompanied by increases in the number of the 23-bp repeats present on the telomeric fragments. These results suggest that either telomerase-maintained telomeres can be more complex in structure than was previously imagined or that Candida telomeres are maintained via a telomerase-independent mechanism.     

Cytogenetics for the model system Arabidopsis thaliana

A detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 microns, which is 20-25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm-ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.     

Fertilization-independent seed development in Arabidopsis thaliana

We report mutants in Arabidopsis thaliana (fertilization-independent seed:fis) in which certain processes of seed development are uncoupled from the double fertilization event that occurs after pollination. These mutants were isolated as ethyl methanesulfonate-induced pseudo-revertants of the pistillata phenotype. Although the pistillata (pi) mutant has short siliques devoid of seed, the fis mutants in the pi background have long siliques containing developing seeds, even though the flowers remain free of pollen. The three fis mutations map to loci on three different chromosomes. In fis1 and fis2 seeds, the autonomous endosperm nuclei are diploid and the endosperm develops to the point of cellularization; the partially developed seeds then atrophy. In these two mutants, proembryos are formed in a low proportion of seeds and do not develop beyond the globular stage. When FIS/fis plants are pollinated by pollen from FIS/FIS plants, approximately 50% of the resulting seeds contain fully developed embryos; these seeds germinate and form viable seedlings (FIS/FIS). The other 50% of seeds shrivel and do not germinate; they contain embryos arrested at the torpedo stage (FIS/fis). In normal sexual reproduction, the products of the FIS genes are likely to play important regulatory roles in the development of seed after fertilization.     

Arabidopsis thaliana mutants altered in homologous recombination

Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information. In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells. We have isolated nine Arabidopsis thaliana mutants hypersensitive to x-ray irradiation (xrs) and examined their recombination properties. For the three xrs loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMCs) and/or methyl methanesulfonate (MMSs) and alterations in homologous recombination. Mutant xrs9 (Xrays, MMSs) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the rad52 epistatic group. xrs11 (Xrays, MMCs) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect. xrs4 (Xrays, MMSs, MMCs) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination. A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination.     

Herbicide safener-inducible gene expression in Arabidopsis thaliana

The potential use of a new chemical-inducible gene expression system in Arabidopsis thaliana has been examined. The system is based on the maize In2-2 promoter which is activated by benzenesulfonamide herbicide safeners. Plants transformed with the beta-glucuronidase (gus) reporter gene under the control of the In2-2 promoter were grown in the presence of different safeners and the induced GUS activity pattern was studied histochemically. In the absence of safeners, the In2-2 promoter was not active. Application of different safeners induced distinct gus expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. Plants maintained continuously on inducing concentrations of the safeners were retarded in growth. The growth inhibition effects of the Sa5 safener could be overcome in a sulfonylurea-resistant background. In2-2 promoter activity could also be induced by the sulfonylurea herbicide chlorsulfuron. In the sulfonylurea-resistant background, which derives from herbicide-resistant acetolactate synthase activity, induction of the In2-2 promoter by chlorsulfuron was lower. Furthermore, branched-chain amino acids, known to inhibit acetolactate synthase activity, also induced In2-2 promoter activity. Our data suggest a strong correlation between In2-2 expression and inhibition of the acetolactate synthase activity.     

Receptor-like protein kinase genes of Arabidopsis thaliana

The isolation of a maize cDNA clone that encodes a membrane spanning protein kinase related to the self-incompatibility glycoproteins (SLG) of Brassica and structurally similar to the growth factor receptor tyrosine kinases has recently been reported. Three distinct receptor-like protein kinase (RLK) cDNA clones from Arabidopsis thaliana have now been identified. Two of the Arabidopsis RLK genes encode SLG-related protein kinases but have different patterns of expression: one is expressed predominantly in rosettes while the other is expressed primarily in roots. The third RLK gene contains an extracellular domain that consists of 21 leucine-rich repeats that are analogous to the leucine-rich repeats found in proteins from humans, flies and yeast. The Arabidopsis leucine-rich gene is expressed at equivalent levels in roots and rosettes. These results show that there are several genes in higher plants that encode members of the receptor protein kinase superfamily. The structural diversity and differential expression of these genes suggest that each plays a distinct and possibly important role in cellular signaling in plants.     

Polyamines and paraquat toxicity in Arabidopsis thaliana

The morbidly obese have a disproportionately greater risk of hypertension, diabetes, and coronary artery disease than their lean or less seriously obese counterparts. Roux-en-Y gastric bypass surgery has been found to be highly effective in inducing, and sustaining, weight loss in individuals with morbid obesity. The purpose of the present study was to examine the effects of weight loss with Roux-en-Y gastric bypass surgery (GBP) on blood pressure, fasting blood glucose, and the lipid/lipoprotein status of 61 morbidly obese women and 21 men. Anthropometric and blood pressure assessments and blood samples for glucose and lipid/lipoprotein analyses were obtained before surgery and at 6 to 12 months postoperatively. By this time, morbidly obese (MO) males and females had lost 33% and 30% of their initial body weight, respectively, along with significant reductions in fasting blood glucose (p < 0.01) and systemic blood pressure (p < 0.05). Weight loss with GBP was also associated with significant reductions in the apoprotein B-containing lipoproteins and the triglyceride and cholesterol composition of these particles. There was a trend (p < 0.10) toward increased serum levels of high density lipoprotein (HDL)-cholesterol following GBP, and significant (p < 0.05) improvement in HDL subfraction distribution and composition. These findings demonstrate the effectiveness of GBP in inducing metabolic changes in the MO population, which may reduce the risk of coronary artery disease, diabetes, and hypertension.     

Differential display of genome subsets containing specific interspersed repeats

A genomic differential display method was developed that analyzes many restriction fragment length polymorphisms simultaneously. Interspersed repeat sequences were used to reduce DNA sample complexity and to target genomic subsets of interest. This work focused on trinucleotide repeats because of their importance in human inherited diseases. Immobilized repeat-containing oligonucleotides were used to capture genomic DNA fragments containing sequences complementary to the oligonucleotide. Captured fragments were amplified by PCR and fluorescently labeled using primers complementary to the repeat sequence and/or to the known sequences ligated to the ends of the restriction fragments. The labeled PCR fragments were displayed by size on a high-resolution automated fluorescent DNA sequencing instrument. Although there was a conservation in the overall pattern of displayed genome subsets, many clear and reproducible differences were detected when genomes from different individuals were compared. Fewer differences were detected within, than between, monozygotic twin pair genomes. In control experiments, the method distinguished between Huntington disease alleles with normal and expanded CAG repeat lengths.     

Cloning and characterization of the Arabidopsis thaliana lupeol synthase gene

A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism. The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product. Lupeol synthase is a multifunctional enzyme that forms other triterpene alcohols, including beta-amyrin, as minor products. Sequence analysis suggests that lupeol synthase diverged from cycloartenol synthase after plants diverged from fungi and animals. This evolutionary order is the reason that fungi and animals do not make lupeol.     

野生型拟南芥叶片愈伤组织诱导的研究

[目的]研究野生型拟南芥叶片愈伤组织的诱导.[方法]以野生型拟南芥叶片为外植体,通过将其切段后接种在添加有不同浓度6-BA和NAA的MS培养基上,研究了拟南芥愈伤组织的诱导和再生植株.[结果]6-BA对于愈伤组织诱导不可缺少,但其浓度太高易产生玻璃化;NAA单独使用利于生根,配合6-BA使用利于分化芽;获得最适愈伤组织诱导培养基为MS+0.50 mg/L 6-BA+0.10mg/L NAA,愈伤组织诱导率可达100%.[结论]为进一步开展拟南芥的遗传转化或细胞培养奠定了基础.     

Targeted disruption of the TGA3 locus in Arabidopsis thaliana

BACKGROUND: Severe latissimus dorsi muscle damage may compromise cardiomyoplasty performance. We analyzed factors underlying the damage produced in 20 sheep latissimus dorsi muscles by isolating the influences of electrical stimulation, mobilization (with some loss of vascular supply), loss of normal resting tension, or a combination of these. METHODS: In group I (n = 3), the muscle was mobilized except for its neurovascular pedicle and reattached at normal resting length. In group II (n = 3), the muscle was mobilized and reattached at about 80% of resting length. Groups III (n = 6) and IV (n = 4) were as groups I and II except that continuous indirect stimulation at 2 Hz was added after 2 weeks. In group V (n = 4), the undisturbed muscle received stimulation alone. After 10 to 12 weeks, muscle samples were taken for morphometric analysis. RESULTS: Loss of resting muscle tension appeared to be the single most damaging intervention, though mobilization and stimulation had further deleterious effects. The worst damage was seen when all three factors were combined, when 60% of the muscle cross section was occupied by connective tissue and fat. The changes were significantly more severe in the distal than in the proximal part of the muscle, implicating ischemia as a contributory factor. CONCLUSIONS: Fiber damage reduces the effectiveness of muscle grafts used for cardiac assistance and merits further systematic investigation.     

Genetic analysis of organ fusion in Arabidopsis thaliana

Postgenital organ fusion occurs most commonly during reproductive development and is important in many angiosperms during genesis of the carpel. Although a number of mutants have been described that manifest ectopic organ fusion, little is known about the genes involved in regulating this process. In this article we describe the characterization of a collection of 29 Arabidopsis mutants showing an organ fusion phenotype. Mapping and complementation analyses revealed that the mutant alleles define nine different loci distributed throughout the Arabidopsis genome. Multiple alleles were isolated for the four complementation groups showing the strongest organ fusion phenotype while the remaining five complementation groups, all of which show only weak floral organ fusion, have a single representative allele. In addition to fusion events between aerial parts of the shoot, some mutants also show abnormal ovule morphology with adjacent ovules joined together at maturity. Many of the fusion mutants isolated have detectable differences in the rate at which chlorophyll can be extracted; however, in one case no difference could be detected between mutant and wild-type plants. In three mutant lines pollen remained unresponsive to contact with the mutant epidermis, demonstrating that organ fusion and pollen growth responses can be genetically separated from one another.     

Cloning of the cDNA for glutamyl-tRNA synthetase from Arabidopsis thaliana

High-sensitivity titration calorimetry is used to measure changes in enthalpy, heat capacity and protonation for the binding of captopril to the angiotensin I-converting enzyme (ACE; EC 3.4.15.1). The affinity of ACE to captopril is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggests that the protonated group in the captopril-ACE complex exhibits a heat protonation of approximately -30 kJ/mol. This value agrees with the protonation of an imidazole group. The residues which may become protonated in the complex could be two histidines existing in two active sites, which are joined to the amino acids coordinated to Zn2+. Calorimetric measurements indicate that captopril binds to two sites in the monomer of ACE, this binding being enthalpically unfavorable and being dominated by a large positive entropy change. Thus, binding is favored by both electrostatic and hydrophobic interactions. The temperature dependence of the free energy of binding deltaG degrees is weak because of the enthalpy-entropy compensation caused by a large heat capacity change, deltaCp =-4.3+/-0.1 kJ/K/mol of monomeric ACE. The strong favorable binding entropy and the negative deltaCp indicate both a large contribution to binding due to hydrophobic effects, which seem to originate from dehydration of the ligand-protein interface, and slight conformational changes in the vicinity of the active sites.     

拟南芥抗氧化突变体筛选条件的建立

[目的]建立筛选拟南芥抗氧化突变体的条件.[方法] 将灭菌的野生型拟南芥种子播种于MS培养基上,4 ℃层化2 d,并于23 ℃培养室中垂直放置培养4 d后,将幼苗转移至含有不同浓度的甲基紫精(MV)胁迫培养基中,倒置培养,比较不同MV浓度下根的弯曲生长情况,以根停止弯曲生长的MV浓度作为抗氧化胁迫突变体的筛选条件.[结果]野生型拟南芥幼苗的根在MV浓度为0.7 μmol/L时受到明显抑制,停止生长;在MV浓度低于0.7 μmol/L时能正常生长或被轻微抑制.[结论]确定筛选抗氧化突变体的MV浓度为0.7 μmol/L.     

Free-flow electrophoresis for fractionation of Arabidopsis thaliana membranes

Highly purified tonoplast and plasma membrane vesicles were isolated from microsomes of Arabidopsis thaliana by preparative free-flow electrophoresis. The most electronegative fractions were identified as tonoplast using nitrate-inhibited Mg2+-ATPase as enzyme marker. The least electronegative fractions were identified as plasma membrane using glucan-synthase II, UDPG: sterol-glucosyl-transferase, and vanadate-inhibited Mg2+-ATPase as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (endoplasmic reticulum) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane. Immunoblot analysis of membrane fractions by antibodies directed against tonoplast and plasma membrane proteins confirmed the nature and the purity of the isolated membranes. The cytoskeletal protein actin, which was also identified by immunoblotting, was found to be specifically attached to the plasma membrane vesicles. The structural and functional integrity of the isolated membranes from Arabidopsis thaliana is discussed in the light of results obtained for the location of receptors and enzymes, or for the determination of ligand binding activity.     

Inflorescence-specific genes from Arabidopsis thaliana encoding glycine-rich proteins

Genomic and cDNA clones for three inflorescence-specific genes from Arabidopsis thaliana were isolated and characterized. The genes are tandemly organized in the genome on a 10 kb fragment. The expression of these genes is coordinately regulated in a developmental and organ-specific pattern. They are expressed predominantly in anthers at the later stage of flower development. The primary structure of the encoded gene products exhibits comparable features consisting of a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Little homology is observed either between the glycine-rich domain of the three genes or with previously described glycine-rich proteins from other plant species.     

The flow karyotype of Arabidopsis thaliana interphase chromosomes

The DNA contents of various aneuploid lines of Arabidopsis thaliana were measured by flow cytometry of 4',6-diamidino-2-phenylindole-stained interphase nuclei in suspensions and compared with each other as well as with the wild-type. The fluorescence intensifies for all lines were highly reproducible as were the deviations from the wild-type. The results allowed the estimation of the relative DNA contents of each Arabidopsis chromosome and of chromosomes arms. The sum of the surplus values for all trisomics was close to the value expected for the haploid (2C) DNA content. Only the line with the smallest telotrisome (Tr 3A) did not significantly differ in DNA content from that of the wild-type. It is concluded that approximately 3% of the genome represents the limit for resolution of differences in DNA content in this system. Thus, the approach allows a fast and reliable screening for duplications and deficiencies extending to 3% of the Arabidopsis genome. Regarding chromosomes sizes a comparison of the flow karyotype with existing karyotypes revealed differences which are discussed.