Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green algaScenedesmus obliquus,mutant C-2A′

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNA^Glu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a K _m value of 2.3 M ± 0.3 for glutamate.Abbreviations ALA 5-aminolevulinic acid - FPLC fast protein liquid chromatography - Glu glutamate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SDS sodium dodecylsulfate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine This work was supported by a grant of the Deutsche Forschungsgemeinschaft. U.C. Vothknecht is grateful for a Nachwuchs-förderungsstipendium des Landes Hessen. The authors want to thank Ms. B. Böhm, J. Gade and K. Eckhardt for skillful technical assistance. The authors also want to thank Dr. C.G. Kannangara (Carlsberg Institute, Kopenhagen, Denmark) for the donation of tRNA from barley and Dr. D. Jahn (FB Biology/Microbiology, Philipps-University, Marburg, FRG) for the tRNA^Glufrom E. coli.     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Inactivation of the streptokinase gene prevents Streptococcus equisimilis H46A from acquiring cell-associated plasmin activity in the presence of plasminogen

Abstract The effects of some physico-chemical parameters on production of extracellular α-L-arabinofuranosidase by Aspergillus nidulans were examined. Highest levels of α-L-arabinofuranosidase were generated with cultures grown on 1% (w/v) purified beet pulp arabinan at 30°C and at an initial pH of 7.0. The enzyme was shown to be very sensitive to the action of proteases. Zymogram overlay of a protein profile obtained by SDS-PAGE revealed the occurrence of a band ( M _r 36 000) exhibiting α-L-arabinofuranosidase activity. The isoelectric pH of the enzyme lay near 4.3. Temperature and pH optima for the activity of crude α-L-arabinofuranosidase preparations were 55°C and 5.5, respectively. Enzyme activity was greatly reduced by thiol reagents such as Hg²⁺ and p -hydroxymercuribenzoate and showed a K _m value of 2.7 mM on p -nitrophenyl α-L-arabinofuranoside as substrate.     

Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement

Summary Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.Abbreviations AM Arabidopsis medium - ANOVA analysis of variance - DAPI 4,6-Diamidino-2-phenylindole - PEG polyethyleneglycol     

Purification and properties of poly(3-hydroxyvaleric acid) depolymerase from Pseudomonas lemoignei

During growth on poly(3-hydroxyvaleric acid), P(3HV), or valerate Pseudomonas lemoignei secretes a P(3HV) depolymerase. This P(3HV) depolymerase was purified from the culture medium of valerate-grown cells by ammonium sulphate precipitation, chromatography on DEAe-sephacel and CM-Sepharose CL 6B. The relative molecular masses of the native as well as the sodium dodecyl sulphate (SDS)-treated enzyme were 53 000 or 54 000, respectively. In contrast to the poly(3-hydroxybutyric acid), P(3HB), depolymerase of Comamonas sp. and P(3HB) depolymerases A and B of P. lemoignei, which are specific for the hydrolysis of P(3HB), the purified P(3HV) depolymerase hydrolysed P(3HB), P(3HV) and co-polymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid at similar rates. Poly(hydroxyalkanoic acids), consisting of monomers with six and more carbon atoms or substrates characteristic for lipases such as Tween 80 or triolein were not hydrolysed. Maximum activities were measured in 50mm TRIS-HCl buffer, pH 8.0, at 55° C. The apparent K _m values of the purified P(3HV) depolymerase for P(3HB) and P(3HV) were 77 and 65 g polyester/ml, respectively. As the main product of enzymatic hydrolysis of P(3HV), 3-hydroxyvalerate was identified. The depolymerase was insensitive to p-hydroxymercuribenzoate but sensitive to dithioerythritol and phenylmethylsulphonyl fluoride, indicating the absence of active reduced sulphur groups and the presence of essential disulphide bonds and serine residues. Correspondence to: D. Jendrossek     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

GENOTYPIC VARIATION AND ALTERNATING DNA LEVELS AT CONSTANT CHROMOSOME NUMBERS IN THE LIFE HISTORY OF THE BROWN ALGA HAPLOSPORA GLOBOSA (TILOPTERIDALES)1

The brown algal order Tilopteridales contains three monospecific genera with reduced life histories, Which are assumed to have been derived form ancestors with oogamous reproduction and alternation of generations. The Newfoundland population of Haplospora globosa Kjellman still shows an alternation of gametophytes and sporophytes, but the chromosome Numbers remain equal because of parthenogenesis and apomeiosis, However, DNA fluorometry showed that the DNA level is twice as high in the Sporophytes as in the gametophytes, The DNA variation at constant chromosome numbers is presumably due to endomitosis combined with a law degree of polyteny. A genotypic variant of Haplospora is represented by the population at Helgoland (F.R.G.) where only sporophytes exist, Spores develop into sporophytes instead of gametophytes, and the plants have reduced chromosome number but the same DNA level as the Newfoundland sporophytes     

A novel type of meso-diaminopimelic acid-based peptidoglycan and novel poly (erythritol phosphate) teichoic acids in cell walls of two coryneform isolates from the surface flora of French cooked cheeses

The primary structure of the peptidoglycan and the teichoic acids of two coryneform isolates from the surface flora of French cooked cheeses, CNRZ 925 and CNRZ 926, have been determined. In the peptidoglycan, meso-diaminopimelic acid was localized in position three of the peptide subunit. It contained an d-glutamyl-d-aspartyl interpeptide bridge, connecting meso-diaminopimelic acid and d-alanine residues of adjacent peptide subunits. The -carboxyl group of d-glutamic acid in position two of peptide subunits was substituted with glycine amide. The teichoic acid pattern and composition differed between the strains: both contained an erythritol teichoic acid and strain CNRZ 925 also contained an N-acetylglucosaminylphosphate polymer. The erythritol teichoic acids differed in terms of the quality and quantity of substituents, but they both had N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid in common.Abbreviations DNP dinitrophenyl - Ery erythritol - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - GlcUANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid - Hex UANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyhexuronic - acid m-Dpm, meso-diaminopimelic acid - Mur muramic acid - MurNAc N-acetylmuramic acid     

Age-related nonrandom chromosomal abnormalities in human low-grade astrocytomas

We report a cytogenetic investigation of 55 low-grade astrocytomas in 52 patients, 15 children and 37 adults. In addition to numerical aberrations such as trisomy 7 and gonosomal losses, we found structural and/or numerical aberrations of chromosome 1 in eight astrocytomas. There was a striking difference between the rearranged chromosomes in pediatric and adult patients. Whereas the pediatric tumors revealed monosomies 1p with accompanying trisomy 1q, the astrocytomas in adults showed partial or complete monosomies 1q.