Molecular basis for determining the sensitivity of eucaryotes to the antimitotic drug rhizoxin

Summary Rhizoxin, an antibiotic, exhibits potent anti-mitotic activity against most eucaryotic cells including those of higher vertebrates, plants and fungi by binding to -tubulin. ThebenA gene of three independently isolated rhizoxin-resistant (Rhi^r) mutants ofAspergillus nidulans was cloned, sequenced and compared with that of the wild-type, rhizoxin-sensitive (Rhi^s) strain. In all three Rhi^r mutants, the AAC codon for Asn-100 of thebenA -tubulin gene was altered to ATC, coding for Ile. Sequence displacement experiments confirmed that the substitution of Ile for Asn-100 confers resistance to rhizoxin in this organism. The amino acid sequences of -tubulin surrounding the 100th amino acid residue from the N-terminus including Asn-100 are highly conserved with a few exceptions. The fission yeastSchizosaccharomyces pombe and the budding yeastSaccharomyces cerevisiae are naturally occurring Rhi^r organisms whose -tubulin genes encode Ile and Val respectively at the 100th amino acid residue. The Ile-100 ofS. pombe and the Val-100 ofS. cerevisiae were altered to Asn using site-directed mutagenesis and gene displacement techniques. The resultant haploid strains of these two yeasts uniquely expressing -tubulin (Asn-100) instead of -tubulin (Ile-100 or Val-100) were found to be Rhi^s. Haploid yeast expressing -tubulin (Asn-100) is normal except for its sensitivity to rhizoxin. These results suggest that rhizoxin resistance has a common basis in both naturally occurring species and experimentally selected mutants in the substitution of Ile or Val for Asn-100 in -tubulin.     

The β-tubulin gene family evolution in theDrosophila montium subgroup of themelanogaster species group

The 1-, 2-, and 3-tubulin genes have been mapped by in situ hybridization on the polytene chromosomes of 11 selected species (15 strains) belonging to theDrosophila montium subgroup. Although the hybridization pattern among the strains of the same species does not differ, this pattern is significantly different among the species. The -tubulin genes in themontium subgroup seem to be organized in a cluster, or in a semi-cluster, or are completely dispersed. The clustered arrangement is found in the North-Oriental sibling speciesD. auraria, D. triauraria, andD. quadraria. The semi-clustered arrangement, wherein the 1 and 2 genes are located at the same locus while 3 is at a different one, appears in the South-Oriental speciesD. bicomuta, D. serrata, andD. birchii, as well as in the Afrotropical speciesD. diplacantha andD. seguyi. The complete separation of the genes is observed in the Indian speciesD. kikkawai andD. jambulina and in the Afrotropical speciesD. vulcana. Based on the above results, a possible mode of evolution of the -tubulin genes in the montium subgroup is attempted. In addition, phylogenetic relationships among themontium species are discussed. Correspondence to: Z.G. Scouras     

Metal resistance in Acinetobacter and its relation to β-lactamase production

Thirty nine clinical isolates of Acinetobacter belonging to six species were tested for resistance to 20 metal ions and their ability to produce -lactamase. Fifty two percent of the strains produced -lactamase. -Lactamase producers and non-producers were almost equally distributed in the different species. A. baumannii was the predominant biotype and was found to be most resistant to metals. Resistance to mercury was prevalent in -lactamase-producing A. baumannii only. Silver resistant strains of A. baumannii produced -lactamase. Sensitivity and resistance to copper and cadium was equally distributed between -lactamase producers and non-producers. -Lactamase-producer and -non-producer strains were uniformly sensitive to cadmium except Acinetobacter genospecies 1.     

Cell-cycle-related variation in proteins in suspension-cultured rice cells

To understand the cell cycle process in plants, we searched for proteins that quantitatively change during the cell cycle in suspension-cultured rice (Oryza sativa L.) cells. The proteins were analyzed by a two-dimensional polyacrylamide gel electrophoresis image-analysis system. We detected 11 proteins that quantitatively changed during the cell cycle, among which -tubulins and a calreticulin-like protein were identified. The amounts of -tubulin proteins were low in the M phase and high in the G1 phase. In contrast, mRNAs for two of the three types of -tubulin were high in the M phase of the cell cycle. The addition of protease inhibitors MG132 or E64d to the cells decreased the -tubulin proteins during 24 h, suggesting that -tubulin proteins are degraded in vivo by proteases other than those whose activities are inhibited by MG132 or E64d.     

Immunological homologues of theArabidopsis thaliana β1 tubulin are polyglutamylated inNicotiana tabacum

Summary Peptide-specific antibody AAB1, raised to the C-terminal 13 amino acids ofArabidopsis thaliana 1 tubulin, identifies a single electrophoretically separable -tubulin on 2-D-gel Western blots of total protein extracts fromA. thaliana seedlings. We show that AAB1 crossreacts with two of the eight polyglutamylated -tubulin isoforms present in purifiedNicotiana tabacum tubulin fractionated by high-resolution isoelectric focussing. Immunolocalisation studies using AAB1 revealed that the twoN. tabacum polyglutamylated 1-tubulin isoforms are utilised in all four plant microtubule arrays (the interphase cortical array, the preprophase band, the spindle and the phragmoplast) indicating that there is no apparent subcellular sorting of these isotypes.Abbreviations AAB1 Anti-Ambidopsis thaliana 1-tubulin antibody - HRIF high-resolution isoelectric focussing     

Organization and structure of Volvox beta-tubulin genes

Summary Genomic clones encoding two Volvox -tubulin genes have been isolated and shown to represent the only two -tubulin genes in the genome. Restriction fragment length polymorphism analysis was used to demonstrate that the two genes are genetically linked. One of these genes was sequenced and the mRNA start site(s) determined by primer extension. A comparison of its sequence to those of the two -tubulin genes of Chlamydomonas revealed: (1) a high degree of conservation of the coding region, with the predicted amino acid sequence differing only in the C-terminal residue; (2) extensive sequence conservation in the 5 untranslated leader region and a 16 bp (putative regulatory) sequence in the promoter region; (3) the same number and location of introns, with a short region of homology in intron 1, but little significant homology in introns 2 and 3.     

Isozyme polymorphism ofβ-glucosidase inAspergillus nidulans

Electrophoretic analysis of the distribution of various electromorphs at different -glucosidase zones was carried out in natural populations ofA. nidulans, theA. nidulans group, and various species belonging to the genusAspergillus from diverse geographical areas of India. The data show the existence of three segregating zones for -glucosidase, designated -GluI, -GluII and -GluIII. All three zones are present in wild isolates ofA. nidulans, and only two, i.e., -GluI and -GluIII, in theA. nidulans group and -GluII and -GluIII in different species ofAspergillus exceptA. terreus, A. flavus, andA. brevipes, where only -GluIII is present. Overall nine electromorphs are observed at -GluI, three at -GluII, and six at -GluIII zones, respectively, It can be concluded that there may be three structural genes for -glucosidase coding the three polymorphic zones inA. nidulans.This research work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi.     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1

In plants, Glycoside Hydrolase (GH) Family 1 -glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thalianagenome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron–exon organizations. The forty-eighth member of this family (At3g06510; sfr2) is a -glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44-encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for -mannosides including 1,4--D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm -mannosidase and barley seed -glucosidase/-mannosidase BGQ60.     

Biosynthesis of cyclic β-(1–3),β-(1–6) glucan in Bradyrhizobium spp.

Inner membranes of Bradyrhizobium japonicum strain USDA 110 produced in vitro soluble and insoluble -(1–3),-(1–6) glucans. The reaction proceeded through a 90 kDa inner membrane intermediate protein; used UDP-glucose as sugar donor and required Mg²⁺. Gel chromatography of soluble glucans resolved a cyclic -(1–3) glucan with a degree of polymerization of eleven from a family of -(1–3),-(1–6) glucans with variable degree of polymerization higher than eleven. Bradyrhizobium strains BR4406 and BR8404 isolated from tree legume nodules in Southeast Brazil produce -(1–3),-(1–6) glucans very similar to that of B. japonicum. A 100 kDa protein was identified in these strains as intermediates in the synthesis of these glucans. Inner membranes of B. japonicum USDA110, B. japonicum I17, and Bradyrhizobium strains BR4406 and BR8404 incubated with UDP-glucose were unable to synthesize -(1–2) glucan and lacked the 235 kDa intermediate protein known to be involved in the synthesis of -(1–2) glucan in Agrobacterium tumefaciens, Rhizobium meliloti and Rhizobium loti.Abbreviations EPS= exopolysaccharides - CPS= capsular polysaccharides - LPS= lipopolysaccharides - AMA= Yeast extract-mannitol medium - TY= tryptone-yeast extract - PMSF= phenyl methyl sulfonil fluoride

---

     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Half-lives of oat mRNAs in vivo and in a polysome-based in-vitro system

We have used a cell-free polysome-based in-vitro mRNA-degradation system to investigate the halflives of plant cell mRNAs. In order to establish the fidelity of the in-vitro system, we used cordycepin to determine the in-vivo half-lives of -tubulin and actin mRNAs in the primary leaves of 4-d-old etiolated oat (Avena sativa L.) seedlings. The in-vitro rank order of half-lives for phytochrome A (45 min), -tubulin (105 min), and actin (220 min) mRNAs mimicked the in-vivo rank order. A pulse of red light given to excised etiolated primary leaves caused an in-vivo reduction in the half-life of -tubulin mRNA. The selectivity of the polysome-based system was further demonstrated by the decrease in the half-life of -tubulin mRNA (from 105 min to 60 min) induced by a pulse of red light given to the etiolated oat seedlings prior to isolation of polysomes. Red light did not affect the apparent half-lives of phytochrome A or actin mRNAs.Abbreviations cab gene for chlorophyll-a/b-binding protein - kb(p) kilobase (pair) - phyA gene for type-I phytochrome protein - rbcS gene for ribulose-1,5-bisphosphate-carboxylase small-subunit We thank Dr. Richard B. Meagher for the pSAc3 actin clone. We thank Dr. Cecil Stewart for the use of his density-gradient fractionator, and Dr. Virginia Crane for instruction in using the fractionator. We also appreciate the helpful comments provided by the other members of the laboratory during the course of this research: Dr. Isaac John, Dr. Iffat Rahim, Linda Barnes, Bruce Held, David Higgs, and Theresa Tirimanne. This work was supported by USDA grants CRGO 88-37261-4196 and 91-37304-6397, and the Iowa State University Biotechnology Program.     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

Molecular characterization of four β-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations. We have previously reported that -tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297–308, 1998). In order to ascertain associations of other tubulins with dinitroaniline resistance, four -tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes. Sequence analysis of the four -tubulin cDNA classes identified no missense mutations. Identified nucleotide substitutions did not result in amino acid replacements. These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast. Expression of the four -tubulins was highest in inflorescences. This is in contrast to -tubulin TUA1 that is expressed predominantly in roots. Collectively, these results imply that -tubulin genes are not associated with dinitroaniline resistance in goosegrass. Phylogenetic analysis of the four -tubulins, together with three -tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.     

Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene

Summary A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial -glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of -glucuronidase activity.     

Mannosyl-β(1-4)-N-acetylglucosaminyl-β(1-N)-urea,a compound isolated from the urine of patients with β-mannosidosis

A previously unknown substance, mannosyl-(1–4)-N-acetylglucosaminyl-(1-N)-urea, has been isolated from the urine of patients with -mannosidosis in addition to the main metabolite mannosyl-(1–4)-N-acetylglucosamine. Structural investigation was carried out by fast atom bombardment mass spectrometry and high-resolution¹H-nuclear magnetic resonance spectroscopy at 500 MHz. It was postulated that the occurrence of this carbohydrate-urea conjugate in urine results mainly from urine handling.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Several phenotypic changes in the cell envelope of Agrobacterium tumefaciens chvB mutants are prevented by calcium limitation

The chvB gene of Agrobacterium tumefaciens encodes a 235 kDa proteinaceous intermediate involved in the synthesis of -1,2-glucan. chvB mutants show a pleiotropic phenotype. Besides not to produce cyclic -1,2-glucan, chvB mutants have been reported to be avirulent, attachment-deficient, and nonmotile. In this study we report additional differences from the parent strain, probably all linked to changes in the cell envelope. This pleiotropic phenotype — except for attachment and virulence — could largely be prevented by growing chvB cells with low levels of calcium. Although a role for -1,2-glucan in osmoadaptation has been proposed, the mode of action of -1,2-glucan is not known. We speculate that in A. tumefaciens -1,2-glucan stabilizes membranes, which would be important especially in hypotonic media containing calcium.Abbreviations Cb carbenicillin - Km kanamycin - TCA trichloroacetic acid - K_av fraction of the stationary gel volume available for diffusion - LPS lipopolysaccharide - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis