Characterization of two beta-tubulin genes from Geotrichum candidum.

Summary The -tubulin genes G1 and G2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G1 and G2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G1 is similar to other fungal -tubulin genes, but G2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5 splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G2. G1 has four introns which are located similarly to those of -tubulin genes in other fungi. G2, however, has a single intron in a unique location. Translational fusions employing the 5 non-coding regions of the two Geotrichum -tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.     

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.     

Elimination of by-products in 11β-hydroxylation of Substance S using Curvularia lunata clones regenerated from NTG-treated protoplasts

Summary Stable mutants showing improved 11-hydroxylation of Substance S were isolated, following treatment with N-methyl-N-nitro-N-nitrosuguanidine (NTG) and regeneration of uninucleate protoplasts of the appropriate fungal strains. This procedure was especially suitable for obtaining more directed 11-hydroxylation of Substance S with Curvularia lunata IM 2901. Apart from producing cortisol (11-hydroxy-S), the parent strain formed several by-products that significantly lowered the yield of the desired 11-hydroxyderivative. Isolated mutants of this microoraganism carried out directed 11-hydroxylation with only a small amount of one of the by-products, which resulted in a much higher yield of cortisol. Correspondence to: L. Sedlaczek     

Beauveria bassiana protoplast regeneration and transformation using electroporation

Summary Regeneration of Beauveria bassiana GK2016 protoplasts demonstrated three phases: (1) enlargement, (2) formation of a chain of budding cells, and (3) development of a true germ tube from the original protoplast. Regeneration frequencies of up to 80% were obtained when using the stabilizer ammonium sulphate. Using electroporation, protoplasts were transformed to methyl 1,2-benzimidazole carbamate (MBC) resistance with the Neurospora crassa MBC-resistant -tubulin gene. A transformation efficiency of one of three transformants per 5 g vector DNA was obtained. The MBC phenotype was stable and transformants grew in the presence of 5 g MBC/ml. Southern DNA-DNA hybridization analysis demonstrated that integration of the vector into the chromosomal DNA had occurred. Correspondence to: G. G. Khachatourians     

Stabilization of steroid 11-hydroxylation activity ofCunninghamella elegans protoplasts in organic osmotic stabilizers

Protoplasts ofCunninghamella elegans, showing 11-, and 11-hydroxylating ability of Substance S, preserved high transformation activity when dispersed in glucose-enriched, organic osmotic stabilizers. A joint action of polyoxins and 2-deoxy-d-glucose was necessary to prevent regeneration of the cell wall in long-lasting experiments. Stabilized and active, dispersed protoplasts may be an alternative research model for studying the function of the cell wall and intracellular metabolic pool constituents in steroid hydroxylation.     

Limited DNA elimination from the irradiated potato parent in fusion products of albino Lycopersicon esculentum and Solanum tuberosum

Summary This paper describes the analysis of the elimination of potato DNA from potato-tomato somatic cell hybrids. The hybrids were obtained by fusion of protoplasts of a cytoplasmic albino tomato genotype with leaf mesophyll protoplasts of a potato genotype carrying the -glucuronidase (GUS) gene of Escherichia coli. The potato protoplasts were either isolated from unirradiated plants or from plants irradiated with 50 or 500 Gy of -rays. Green calli were selected as putative fusion products. The hybridity of these calli was confirmed by isoenzyme analysis. All of the green calli tested contained a potato-specific chloroplast DNA restriction fragment, and most of the calli analysed were positive for -glucuronidase activity. In 72 of the hybrid calli we determined the percentage of potato nuclear DNA using species-specific probes. All of the tested green calli contained a considerable amount of potato genomic DNA, irrespective of the dose of irradiation of the potato parent. The limited degree of potato DNA elimination and the absence of true cybrids are discussed.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Involvement of the cell wall ofNocardia restricta on the bioconversions of steroid sapogenins

The bioconversion of tigogenin, (25R)-5-spirostan-3-ol byNocardia restricta to (25R)-4-spirosten-3-one and (25R)-1,4-spirostadien-3-one was strongly increased using protoplasts instead of intact cells. The same effect could be achieved by adding-cyclodextrin to the medium. Since 5-steroid sapogenins pass through the cell wall without hindrance, it can be concluded that the cell wall ofNocardia restricta is a rate-limiting factor only for 5-isomers.     

The monosaccharide binding site of lentil lectin: an X-ray and molecular modelling study

The X-ray crystal structure of lentil lectin in complex with -d-glucopyranose has been determined by molecular replacement and refined to anR-value of 0.20 at 3.0 Å resolution. The glucose interacts with the protein in a manner similar to that found in the mannose complexes of concanavalin A, pea lectin and isolectin I fromLathyrus ochrus. The complex is stabilized by a network of hydrogen bonds involving the carbohydrate oxygens O6, O4, O3 and O5. In addition, the -d-glucopyranose residue makes van der Waals contacts with the protein, involving the phenyl ring of Phe123. The overall structure of lentil lectin, at this resolution, does not differ significantly from the highly refined structures of the uncomplexed lectin.Molecular docking studies were performed with mannose and its 2-O and 3-O-m-nitro-benzyl derivatives to explain their high affinity binding. The interactions of the modelled mannose with lentil lectin agree well with those observed experimentally for the protein-carbohydrate complex. The highly flexible Me-2-O-(m-nitro-benzyl)--d-mannopyranoside and Me-3-O-(m-nitro-benzyl)--d-mannopyranoside become conformationally restricted upon binding to lentil lectin. For best orientations of the two substrates in the combining site, the loss of entropy is accompanied by the formation of a strong hydrogen bond between the nitro group and one amino acid, Gly97 and Asn125, respectively, along with the establishment of van der Waals interactions between the benzyl group and the aromatic amino acids Tyr100 and Trp128.RL and FC are joint first authors.     

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca²⁺concentration was lowered by ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.Abbreviations ConA concanavalin A - CW calcofluor white - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - -Man -methyl-d-mannoside     

Significance of bacterial ectoenzymes in aquatic environments

The report presents studies on temporal and spatial variations of kinetics (Vmax and Km) of bacterial ectoenzyme activity (-glucosidase - Glc, leucine aminopeptidase - Leu-amp) in the naturally eutrophic Plusee. Glc and Leu-amp activity were positively correlated with the flux of polymeric materials (polysaccharides, proteins) in the lake. Glc activity was low when algal populations grew actively, but during the algal bloom breakdown Glc activity increased rapidly. Leu-amp displayed the highest rates of activity in the epilimnion and was tightly coupled to bacterial production. The synthesis of studied ectoenzymes was under control of a repression/derepression mechanism. The significance of ectoenzymes for the transformation and bacterial utilization of organic matter, and their role in the microbial loop in aquatic environments is discussed.     

Low temperature protocol for efficient transformation ofMycobacterium smegmatis spheroplasts

Spheroplasts ofMycobacterium smegmatis LM15, strain 607, were prepared by a combined treatment with glycine and lysozyme. The spheroplasts were tested for ability to take up and express purified mycobacteriophage DNA. Exposure of 1.0×10⁸ to 1.0×10⁹ spheroplasts to saturing DNA (1 g) for 15 min at 5°C resulted in a transfection efficiency of approximately 0.009%. The transfer of the -lactamase marker with DNA purified from strain LM15 to spheroplasts of a -lactamase-negative mutant, strain LM144, was achieved. The DNA-treated cultures, after reversion to the bacillary form, contained 20-fold more penicillinresistant cells than the nontreated control culture. Approximately 80% of the penicillin-resistant colonies from the DNA-treated cells were positive for -lactamase. Cell-free extracts of penicillin-resistant transformants contained -lactamase activity that ranged from 0.046 to 0.134 mol of benzylpencillin hydrolyzed/min per mg protein. This low temperature procedure is recommended for high efficiency transformation ofM. smegmatis.     

The mechanism of object fixation and its relation to spontaneous pattern preferences in Drosophila melanogaster

The orientation behavior of walking flies, Drosophila melanogaster, towards a single 6° wide black vertical stripe (elementary stripe) can be explained by use of the turning tendency function H(). This function is characterized by maximal values at an angular distance of =25° from the stable zero position (=orienting direction), a sharp decline from this maximum to =60°, and a very slow approach to the unstable zero position (Horn and Wehner, 1975). The shape of this function is influenced by both translatory and rotatory components of movement. If the translatory component is minimized by measuring the turning function W() (see 2.3) at a distance of 10 mm (C1) from the center of the arena, a change in the strength of this decline is caused. But with increasing translatory component, i.e. at a greater distance from the center of the arena, W() approximates the heuristical function H() (Fig. 12). The turning functions W() are pattern-specific; the angular positions of the maximum responses shift to greater angles with increasing width of the patterns (Fig. 2). In the twopattern configuration with double or single stripes, there is always a coincidence between the stable zero positions of W (), the mean of the frequency distributions P() of the flies' positions and n _g() of the straight courses, and the stable zero positions of H () obtained from an additive superposition of two or more angular shifted turning tendency functions H() (Fig. 5, 7). Therefore, the mean positions of the flies in a multi-stripe experiment composed of elementary stripes can be predicted from the addition of many angular shifted turning tendency functions H(). Between H() and the frequency distribution P() of the flies' positions , the following formula holds: P() =C·H()d (Fig. 13). With this equation, the spontaneous preference of the broader of two double stripes can be explained presuming lateral interactions between the components of the patterns (Fig. 8, 10). The strength x _i ^* of this lateral interaction depends on the width of the double stripes. The greater , the smaller is x _i ^* . x _i ^* is a pattern-specific value (Table 1, 2).Supported by the Deutsche Forschungsgemeinschaft, Ho 664/2     

Direct regeneration in vitro and transient GUS expression in Mentha×piperita

Genetic transformation of peppermint is known to be very difficult essentially because of low efficiency regeneration. A regeneration protocol allowing 51% shooting frequency is proposed. Transient -glucuronidase expression and adjustment of selection pressure with kanamycin are also reported. The final retained method to attempt peppermint transformation is:Agrobacterium inoculation or biolistic treatment of the first apical leaves ofin vitro clones, regeneration in the dark with kanamycin (1 mg l^–1) and 6-benzylaminopurine (2 mg l^–1), followed by selection of regenerated shoots with 200 mg 1^–1 kanamycin.Abbreviations BA 6-benzylaminopurine - GUS -glucuronidase - MS Murashige and Skoog (1962) - NAA -naphthalenacetic acid - PIG particle inflow gun - SEM scanning electron microscope     

Genetic control of β-diketone and hydroxy-β-diketone synthesis in epicuticular waxes of barley

Summary Five eceriferum, (cer) mutants in barley which influence -diketone and hydroxy--diketone synthesis in spike and internode epicuticular waxes have been characterized. The mutation cer-u ⁶⁹ blocks the synthesis of hydroxy--diketones and leads to a compensatory increase in the amount of -diketones, indicating that -diketones are precursors of the hydroxy--diketones. Furthermore, highly lobed wax plates were observed for the first time on barley lemmas, in addition to the characteristic wax tubes. Both diketone classes are selectively and proportionally reduced in the spike wax of cer-i ¹⁶, which has shorter wax tubes. The three mutants cer-c ³⁶, -q ⁴², and -c,u ¹⁰⁸ synthesize neither diketone class and form no wax tubes. In contrast to the variable composition of most individual barley wax classes, only a single -diketone was identified, namely hentriacontan-14,16-dione.     

The locus controlling liver GM1(NeuGc) expression is mapped 1 cM centromeric to H-2K

The polymorphic variation of liver GM1 (NeuGc) ganglioside was found in inbred strains of the mouse. The genetic analysis using C57BL/10 (GM1-negative) and SWR (GM1-positive) mice revealed that a single autosomal gene (Ggm-1) was involved in the expression of liver GM1(NeuGc) and that C57BL/10 mice lacking GM1(NeuGc) expression carried a defective gene on Ggm-1. Since our previous study on H-2 congenic mice indicated that Ggm-1 was linked to the H-2 complex, in this study we measured recombination frequencies among Ggm-1, Go-1 and H-2K in the backcross progeny between (C57BL/10 × SWR)F₁ and C57BL/10. Ggm-1 was mapped 1 cM centromeric to H-2K on chromosome 17.Abbreviations used in this paper GM1(NeuGc) Gal1-3GalNAc1-4 (NeuGc2-3)Gal1-4Glc1-ceramide - GM2(NeuGc) Gal1-4(Neu Gc2-3)Gal1-4Glc1-ceramide - GM3(NeuGc) NeuGc2-3Gal1-4 Glc1-ceramide - GD1a(NeuGc) NeuGc2-3Gal1-3GalNAc1-4 (NeuGc2-3)Gal1-4Glc1-ceramide