High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase

A full-length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc Natl Acad Sci USA 86: 2582-2586) driven by the CaMV 35S promoter was introduced into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV 35S-TDC transformed plants had from four to 45 times greater TDC activity than did controls. Tryptamine accumulated in transgenic plants to levels that were directly proportional to their TDC specific activity. Despite their increased tryptamine content, the growth and development of the CaMV 35S-TDC plants appeared normal with no significant differences in indole-3-acetic acid levels between high tryptamine and control plants. Plants with the highest TDC activity contained more than 1 milligram of tryptamine per gram fresh weight, a 260-fold increase over controls.     

The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes. Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization. In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato. We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs. These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated. Comparison to two known yeast MARs ARS1 and CENIII, showed that the HSC80 5MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed. Competition binding between the two HSC80 MARs indicated that the 5 MAR can outcompete the 3 MAR and not vice versa. Last, we observed that the interaction of the 3 MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment. In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene. The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.     

Stratigraphy,facies and synsedimentary tectonics in the Jurassic Rosso Ammonitico Veronese (Altopiano di Asiago,NE Italy)

Summary The red, pelagic limestones of the Rosso Ammonitico Veronese (Upper Bajocian-Tithonian) of the Altopiano di Asiago area can be subdivided into eight facies. They differ from each other in structure (bedding style, presence and type of nodularity) and texture (nature of components, grain-vs mud-support, compactional features). Several discontinuities could be recognized, based on sedimentological or biostratigraphic evidence. In the context of a drowned platform, where sediments essentially consist of skeletal remains of both planktonic and benthic organism, the different facies are interpreted as reflecting a varying influence of currents on the winnowing of micrite and on triggering early cementation. Early cementation in turn, controlled the patterns of bioturbation and the response of sediments to later compaction and pressure-dissolution. At times, microbial mats, of unidentified nature, were important in trapping and binding sediment, giving rise to early lithified nodules and layers of stromatolitic aspect. The Rosso Ammonitico Veronese can be subdivided into three units: lower Rosso Ammonitico (RAI: Upper Bajocian-Lower Callovian), middle Rosso Ammonitico (RAM: Upper Callovian-Middle Oxfordian), and upper Rosso Ammonitico (RAS: Lower Kimmeridgian-Tithonian). Frequent ammonite moulds allow the precise dating of the base and top of each unit, and the documentation of facies heteropies and hiatusses in the more fossiliferous RAS. The lower unit (RAM) is massive and essentially nodular; the middle unit (RAM) is well bedded, non-nodular, and cherty; the upper unit (RAS) is richer in clay and typically nodular. The RAI and the RAS are present everywhere, though significant facies and thickness changes affect particularly the RAS; the RAM is much more variable, ranging from 0 to 10 metres. These variations, that may be gradual or abrupt, are inter-preted as the result of Middle-Late Callovian block-faulting which generated an irregular sea floor topography where the swells were more exposed to currents that continuously removed sediments, inducing long-lasting periods of nondeposition. Sediments preferentially accumulated in the adjacent lows. A confirmation of this hypothesis is provided by evidence of synsedimentary tectonics, described for the first time in the Rosso Ammonitico Veronese. Neptunian dykes, both vertical and horizontal, are developed at the top of the RAI and are filled with laminated sediments or collapse breccias. Glides of metre-size blocks and slumps are present at the top of the RAI and at the base of the RAM, respectively. Cm-thick layers of mud supported breccias are intercalated in the upper part of the RAI and within the RAM: they are interpreted as seismites. All these features document a tensional regime that generated fractures in more or less lithified sediments and failure along steep fault scarps or gently dipping slopes of tilted fault blocks. Recognition of this Callovian-Oxfordian tectonic activity shows that, after the Bajocian drowning, the Trento Plateau did not simply experience a uniform and general foundering: a small-scale block-faulting was still active and affected the pattern of facies distribution.     

Intra- and interobserver concordance in scoring Harris lines: A test on bone sections and radiographs

Little attention has been devoted to assessing the reproducibility of (paleo)pathological observations. Harris lines (HL) are among the markers most used to determine chronology of stresses suffered during growth. Nevertheless, their scoring entails remarkable methodological difficulty. Bone sections (S) and radiographs (R) of 29 adult tibiae of archeological provenance (medieval) were scored for HL by five observers. At regular intervals of time, each observer gave two independent counts on both series. Results show a) a substantial interobserver disagreement of HL estimates for both sectional and radiographic records, and b) a high level of intraobserver error. © 1994 Wiley-Liss, Inc.     

CHROMOSOMAL VERSUS MITOCHONDRIAL DNA EVOLUTION: TRACKING THE EVOLUTIONARY HISTORY OF THE SOUTHWESTERN EUROPEAN POPULATIONS OF THE SOREX ARANEUS GROUP (MAMMALIA,INSECTIVORA)

The shrews of the Sorex araneus group have undergone a spectacular chromosome evolution. The karyotype of Sorex granarius is generally considered ancestral to those of Sorex coronatus and S. araneus. However, a sequence of 777 base pairs of the cytochrome b gene of the mitochondrial DNA (mtDNA) produces a quite different picture: S. granarius is closely related to the populations of S. araneus from the Pyrenees and from the northwestern Alps, whereas S. coronatus and S. araneus from Italy and the southern Alps represent two well-separated lineages. It is suggested that mtDNA and chromosomal evolution are in this case largely independant processes. Whereas mtDNA haplotypes are closely linked to the geographical history of the populations, chromosomal mutations were probably transmitted from one population to another. Available data suggest that the impressive chromosome polymorphism of this group is quite a recent phenomenon.     

A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: characterization,purification and kinetic properties

The enzyme, desacetoxyvindoline 4-hydroxylase, was purified to apparent homogeneity from Catharanthus roseus by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, green 19-agarose, hydroxylapatite, -kg sepharose and Mono Q. The 4-hydroxylase was characterized by its strict specificity for position 4 of desacetoxyvindoline suggesting it to catalyze the second to last step in vindoline biosynthesis. The molecular mass of the native and denatured 4-hydroxylase was 45 kDa and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pIs 4.6, 4.7 and 4.8. The purified 4-hydroxylase exhibited no requirement for divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The enzyme was not inhibited by EDTA or SH-group reagents at concentrations up to 10 mM. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where -kg is the first substrate to bind followed by the binding of O₂ and desacetoxyvindoline. Their K _m values for -kg, O₂ and desacetoxyvindoline are 45 M, 45 M and 0.03 M, respectively. The first product to be released was deacetylvindoline followed by CO₂ and succinate, respectively.Abbreviations -kg -ketoglutarate or 2-oxoglutarate - NMT N-methyltransferase - SAM S-adenosyl-l-methionine - TLC thin layer chromatography - VBL vinblastine - VCR vincristine