Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

Anchorage-independent growth of RSV-transformed rat (GCA, W 12 and XC) cells

Three RSV-transformed rat cell lines: GCA, W12 and XC were characterized as to their ability to anchorage-independent growth in comparison to normal rat kidney (NRK-49F) cells. Differences in the threshold density (TD) and colony forming efficiency (CFE) of the investigated cells are described. The ability of virally transformed cells to stimulation of soft agar colony formation of NRK cells in coculture assay was presented. The production of TGFs-like factors by GCA, W12 and XC cells was suggested.     

Continuous microbial desulfurization of coal--application of a multistage slurry reactor and analysis of the interactions of microbial and chemical kinetics

Microbial desulfurization of coal by pyrite oxidizing bacterial enrichment cultures has been studied in air-agitated slurry reactors of 4- and 20-L volumes. Batch experiments showed that inoculation with an active bacterial culture is essential to minimize the lag phase, although a considerable number of pyrite oxidizing bacteria was found on the coal prior to desulfurization. For detailed investigations of kinetics, energy requirements, and technical applicability, a bioreactor equipment consisting of a cascade of eight stages was developed and operated continuously. Microbial desulfurization of coal-monitored by measuring the axial profile of dissolved iron concentration, real and maximum oxygen consumption rates, and cell concentration-at pulp densities to 30% was performed over a period of 200 days without any disturbances concerning the aeration system, fluidization, transport of solids and microbial growth. At a pulp density of 20%, a pyrite conversion of 68% was achieved after the third reactor stage at a total residence time of five days in the first three stages. The kinetics of pyrite degradation were found to be well described by a rate equation of first order in pyrite surface area concentration if the pyrite is directly accessible for microbial attack. Rate constants were determined to 0.48 mg pyrite/(cm(2) day) in the first and to 0.24 mg pyrite/(cm(2) day) in the following reactor stages. Kinetic models taking into account adsorption/desorption as well as growth kinetics failed to describe the observed reaction rates. However, a model treating pyrite degradation and microbial growth kinetics formalistically seems to be applicable when backmixing between the reactor stages can be avoided. The advantage of a multistage reactor in comparison to single-stage equipment was shown by calculation. To obtain a pyrite conversion of 68%, a three-stage reactor would require only 58% of the volume of single-stage equipment.Measurement of oxygen consumption rates proved to provide quickly and easily measurable parameters to observe microbial coal desulfurization in technical scale: the real oxygen consumption rate is correlated to the pyrite oxidation rate and the maximum oxygen consumption rate is correlated to the concentration of viable cells. The Y(o/s) coefficient for the amount of oxygen consumed per mass unit of pyrite oxygen was determined to approximately 0.33 in comparison to 1.0 which can be calculated from stoichiornetry. This could yet not be explained. Chemical leaching experiments as well as sulfur analyses of desulfurized coal samples showed that the microorganisms play the main role in degradation of pyrite from coal and that pyrite oxidation by ferric iron can be neglected.     

Rat pineal S-antigen: sequence analysis reveals presence of alpha-transducin homologous sequence

S-antigen (S-Ag) is a soluble, highly antigenic protein, the administration of which induces autoimmune uveitis. This protein is found in the retina and pineal. Retinal S-Ag from three species has been sequenced. In this study rat pineal S-Ag was sequenced. Clones were isolated from a rat pineal lambda gt11 cDNA library by probing with a 300 bp fragment of mouse retinal S-Ag cDNA containing the 5'-coding region. The largest clone isolated (RPS-118; 1364 bp) contained the entire coding sequence. Comparison of the rat pineal and mouse retinal S-Ag nucleotide sequences indicated a high homology (95%). The deduced amino acid sequence was found to contain 403 residues (congruent to 44 992 Da). Comparison of the rat pineal and mouse retinal S-Ag amino acid sequences also revealed high homology (97%). The similarity of both the nucleotide and amino acid sequences of rat pineal and mouse retinal S-Ag indicates that expression of the S-Ag gene in both tissues is similar. Further analysis of the rat pineal S-Ag sequence indicated that it contained essentially the same major uveitopathogenic region of S-Ag present in bovine retina; minor uveitopathogenic sites were somewhat different. As is true of retinal S-Ag, rat pineal S-Ag contains the same consensus phosphoryl-binding site present in many GTP/GDP-binding proteins and a homologous sequence found in the C-terminus of alpha-transducin. These sequences may play a role in the action of pineal S-Ag in transmembrane signal transduction.     

Yeast Intrachromosomal Recombination: Long Gene Conversion Tracts Are Preferentially Associated with Reciprocal Exchange and Require the Rad1 and Rad3 Gene Products

A yeast intrachromosomal recombination system based on an inverted repeat has been designed to examine mitotic gene conversion tract length and the association of crossing over with gene conversion as a function of the conversion tract length. Short conversion tracts are found to be preferentially noncrossover while conversion tracts longer than 1.16 kb show a 50% association with crossover. Mutation in the excision repair gene RAD1 leads to a reduction in conversion tracts of at least 1.16 kb and a reduction in crossovers associated with conversion, regardless of the length of the conversion tract. Mutation in the excision repair gene RAD3, which encodes a DNA helicase, also leads to a reduction in conversion tracts of at least 1.16 kb, but has no effect on the frequency of associated crossovers. The roles of RAD1 and RAD3 in recombination are discussed.     

Effects of phospholipases,proteases and neuraminidase on γ-hydroxybutyrate binding sites

Summary -Hydroxybutyric acid (GHB) is a natural compound of mammalian brain synthesized from GABA. The characteristics of its synthesis, transport, release, distribution and turnover, in addition to the presence of a high affinity binding site for this substance in brain are in favor of a modulator role for GHB. The effects of hydrolytic enzymes on the specific binding capacity of GHB have been studied in the present work. Phospholipases A₂ and C, neuraminidase and Pronase markedly decrease GHB binding to crude synaptosomal membranes from rat brain. This effect is time and enzyme concentration dependent. Trypsin, under the conditions employed, is less active. The inhibitory effects of phospholipases is correlated with phospholipid hydrolysis. Lysophospholipids, in the absence of bovine fatty acid free serum albumin partially inhibit GHB binding. The action of neuraminidase has been followed by sialic acid release and modifications of the ganglioside profile. The effects of phospholipase C and of neuraminidase are completely different to those on GABA binding sites. These results represent further data concerning the molecular existence of specific GHB binding sites on rat brain membranes.Abbreviations GHB -hydroxybutyrate - LPC L--lysophosphatidylcholine - LPE Lysophosphatidylethanolamine - PC Phosphatidylcholine - PE Phosphatidylethanolamine - BSA Bovine Serum Albumin     

Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.