The biosynthesis of the lantibiotics epidermin,gallidermin, Pep5 and epilancin K7

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.Abbreviations Dha 2,3-didehydroalanine - Dhb 2,3-didehydrobutyrine - Lan lanthionine - Melan methyllanthionine     

Genetic Analysis of the Epidermal Differentiation Complex (EDC) on Human Chromosome 1q21: Chromosomal Orientation, New Markers, and a 6-Mb YAC Contig

The epidermal differentiation complex (EDC) unites a remarkable number of structurally, functionally, and evolutionarily related genes that play an important role in terminal differentiation of the human epidermis. It is localized within 2.05 Mb of region q21 on human chromosome 1. We have identified and characterized 24 yeast artificial chromosome (YAC) clones by mapping individual EDC genes, sequence-tagged site (STS) markers (D1S305, D1S442, D1S498, D1S1664), and 10 new region-specific probes (D1S3619–D1S3628). Here we present a contig that covers about 6 Mb of 1q21 including the entire EDC. Fluorescencein situhybridization on metaphase chromosomes with two YACs flanking the EDC determined its chromosomal orientation and established, in conjunction with physical mapping results, the following order of genes and STSs: 1cen–D1S442–D1S498–S100A10–THH–FLG–D1S1664–IVL–SPRR3–SPRR1–SPRR2–LOR–S100A9–S100A8–S100A7–S100A6–S100A5–S100A4–S100A3–S100A2–S100A1–D1S305–1qtel. These integrated physical, cytogenetic, and genetic mapping data will be useful for linkage analyses of diseases associated with region 1q21 and for the identification of novel genes and regulatory elements in the EDC.     

Thiophene synthesis and distribution in young developing plants of Tagetes patula and Tagetes erecta

Thiophene synthesis and accumulation were investigated in organsof Tagetes patula and T. erecta. Thiophene accumulation startedrapidly in germinating seedlings of both species. Roots andhypocotyls were the major thiophene accumulating organs and5-(3-buten-1-ynyl)-2, 2-bithienyl (BBT) and 5-(4-acetoxy-1-butynyl)-2,2 -bithienyl (BBTOAc) were the major accumulated compounds.Higher thiophene concentrations were reached in Tagetes patulathan in T. erecta. Accumulation patterns for individual thiopheneswere different within organs, between organs and between bothspecies. Within hypocotyls of Tagetes patula, thiophene concentrationswere high in the epidermis and the vascular tissue and low inthe parenchymatic tissues of cortex and pith. Synthesis of thiopheneswas high in the roots and hypocotyls and very low in the leaves.Transport of thiophenes from the roots into the shoot occurred,but the rate of transport was too low to explain the high concentrationsin the hypocotyl. It is concluded that for the main part thiophenesare accumulated where they are synthesized. Key words: Tagetes, hiophenest, synthesis, accumulation, secondary metabolites     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Light-stimulated proton transport into the vacuoles of leaf mesophyll cells does not require energization by the tonoplast pyrophosphatase

Photosynthetic characteristics of transgenic tobacco (Nicotiana tabacum L.) plants with a soluble pyrophosphatase in the cytosol of their leaf cells were compared to those of wild-type plants. Although the development of the transgenic plants was somewhat retarded compared to the wild type, as shown by stunted growth and delayed flowering, photosynthetic responses were comparable in transgenic and wild-type leaves of similar physiological age. In particular, light-dependent proton transport into the vacuoles of leaf mesophyll cells was not decreased in leaves of the transgenic plants, which did not contain pyrophosphate in the cytosol owing to the presence of a soluble pyrophosphase. This shows that light-stimulated proton pumping did not require the pumping activity of the tonoplast pyrophosphatase. Apparently, light-stimulated proton pumping can be based solely on the activity of the tonoplast ATPase.Abbreviation CDCF 5-(and 6-)arboxy-2,7-dichlorofluorescein This work was supported within the Sonderforschungsbereiche 176 and 251 of the University of Würzburg.     

Low temperature cultivation — A step towards process optimisation

Adherent recombinant BHK cells were cultivated at temperatures between 30 and 37°C. Batch and repeated-batch-cultivations in a 2-litre bioreactor showed a significant influence on metabolism and cell growth. The low-temperature-cultivations showed a lower growth rate and a lower glucose consumption rate and, therefore, less lactate production. On the other hand, the maximum cell density and productivity seemed not to be affected by the temperature reduction.     

Effects of magnetic fields on mammary tumor development induced by 7, 12-dimethylbenz(a)anthracene in rats

A series of epidemiological studies have indicated associations between exposure to magnetic fields (MFs) and a variety of cancers, including breast cancer. In order to test the possibility that MF acts as a cancer promoter or copromoter, four separate experiments have been conducted in rats in which the effects of chronic exposure to MFs on the development of mammary tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) were determined. Female rats were exposed in magnetic coils for 91 days (24 h/day) to either alternating current (AC; 50 Hz)-MF or direct current (DC)-MF. Magnetic flux density of the DC-MF was 15 mT. Two AC-MF exposures used a homogeneous field with a flux density of 30 mT (rms); one used a gradient field with flux density ranging from 0.3–1 μT. DMBA (5 mg) was administered orally at the onset of MF exposure and was repeated thrice at intervals of 1 week. In each experiment, 18–36 animals were exposed in 6 magnetic coils. The same number of rats were used as sham-exposed control. These control animals were treated with DMBA and were placed in dummy coils in the same room as the MF-exposed rats. Furthermore, groups of age-matched rats (reference controls) were treated with DMBA but housed in another room to exclude any MF exposure due to the magnetic stray field from the MF produced by coils. At the end of the exposure or sham-exposure period, tumor number and weight or size of tumors were determined at necropsy. Results were as follows: In sham-exposed animals or reference controls, the tumor incidence varied between 50 and 78% in the 4 experiments. The average number of mammary tumors per tumor-bearing animal varied between 1.6 and 2.9. In none of the experiments did MFs significantly alter tumor incidence, but in one of the experiments with AC-MF exposure at 30 mT, the number of tumors per tumor-bearing animal was significantly increased. Furthermore, exposure to a DC-MF at 15 mT significantly enhanced the tumor weight. Exposure to a gradient AC-MF at 0.3–1 μT exerted no significant effects. These experiments seem to indicate that MFs at high flux densities may act as a promoter or copromoter of breast cancer. However, this interpretation must be considered only a tentative conclusion because of the limitations of this study, particularly the small sample size used for MF exposure and the lack of repetition of data. © 1993 Wiley-Liss. Inc.