Target choice determinants of the Tc1 transposon of Caenorhabditis elegans.

The Tc1 transposon of Caenorhabditis elegans always integrates into the sequence TA, but some TA sites are preferred to others. We investigated a TA target site from the gpa-2 gene of C.elegans that was previously found to be preferred (hot) for Tc1 integration in vivo . This site with its immediate flanks was cloned into a plasmid, and remained hot in vitro , showing that sequences immediately adjacent to the TA dinucleotide determine this target choice. Further deletion mapping and mutagenesis showed that a 4 bp sequence on one side of the TA is sufficient to make a site hot; this sequence nicely fits the previously identified Tc1 consensus sequence for integration. In addition, we found a second type of hot site: this site is only preferred for integration when the target DNA is supercoiled, not when it is relaxed. Excision frequencies were relatively independent of the flanking sequences. The distribution of Tc1 insertions into a plasmid was similar when we used nuclear extracts or purified Tc1 transposase in vitro , showing that the Tc1 transposase is the protein responsible for the target choice.     

Characterization of an endosymbiont infecting wood ticks, Dermacentor andersoni, as a member of the genus Francisella.

A microorganism (Dermacantor andersoni symbiont [DAS]) infecting Rocky Mountain wood ticks (D. andersoni) collected in the Bitterroot Mountains of western Montana was characterized as an endosymbiont belonging to the genus Francisella. Previously described as Wolbachia like, the organism's DNA was amplified from both naturally infected tick ovarial tissues and Vero cell cultures by PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA) and Francisella membrane protein genes. The 16S rDNA gene sequence of the DAS was most similar (95.4%) to that of Francisella tularensis subsp. tularensis. Through a combination of Giménez staining, PCR assay, and restriction fragment length polymorphism analysis, 102 of 108 female ticks collected from 1992 to 1996 were infected. Transovarial transmission to female progeny was 95.6%, but we found no evidence of horizontal transmission.     

Separation of Photolabile-Phytochrome and Photostable-Phytochrome Actions on Growth and Microtubule Orientation in Maize Coleoptiles (A Physiological Approach)

The effect of isoprenoid growth regulators on avocado (Persea americana Mill. cv Hass) fruit growth and mesocarp 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was investigated during the course of fruit ontogeny. Both normal and small-fruit phenotypes were used to probe the interaction between the end products of isoprenoid biosynthesis and the activity of HMGR in the metabolic control of avocado fruit growth. Kinetic analysis of the changes in both cell number and size revealed that growth was limited by cell number in phenotypically small fruit. In small fruit a 70% reduction in microsomal HMGR activity was associated with an increased mesocarp abscisic acid (ABA) concentration. Application of mevastatin, a competitive inhibitor of HMGR, reduced the growth of normal fruit and increased mesocarp ABA concentration. These effects were reversed by co-treatment of fruit with mevalonic acid lactone, isopentenyladenine, or N-(2-chloro-4-pyridyl)-N-phenylurea, but were not significantly affected by either gibberellic acid or stigmasterol. However, stigmasterol appeared to partially restore fruit growth when co-injected with mevastatin in either phase II or III of fruit growth. In vivo application of ABA reduced fruit growth and mesocarp HMGR activity and accelerated fruit abscission, effects that were reversed by co-treatment with isopentenyladenine. Together, these observations indicate that ABA accumulation down-regulates mesocarp HMGR activity and fruit growth, and that in situ cytokinin biosynthesis modulates these effects during phase I of fruit ontogeny, whereas both cytokinins and sterols seem to perform this function during the later phases.     

Interaction of auxin, light, and mechanical stress in orienting microtubules in relation to tropic curvature in the epidermis of maize coleoptiles

Summary Changes in the orientation of cortical microtubules (longitudinal vs. transverse with respect to the long cell axis) at the outer epidermal wall of maize coleoptile segments were induced by auxin, red or blue light, and mechanical stresses (cell extension or compression produced by bending). Immunofluorescent techniques were used for the quantitative determination of frequency distributions of microtubule orientation. Detailed kinetic studies showed that microtubule reorientations are temporally correlated with the simultaneously measured changes in growth rate elicited by auxin, red light, or blue light. Growth inhibition induced by depletion of endogenous auxin produces a longitudinal microtubule pattern that can be changed into a transverse pattern in a dose-dependent manner by applying exogenous auxin. A mid-point pattern with equal frequencies of longitudinal and transverse microtubules was adjusted at 2 mol/1 auxin. Bending stress applied under these conditions adjusts permanent, maximally longitudinal and transverse microtubule orientations at the compressed and extended segment sides, respectively, quantitatively mimicking the responses to differential flank growth during phototropic and gravitropic curvature. During tropic curvature the changes in microtubule pattern reflect the distribution of growth rather than the distribution of auxin. The microtubule pattern responds to auxin-dependent growth changes and mechanical stress in a synergistic manner, confirming the functional equivalence of these factors in affecting microtubule orientation. Similar results were obtained when segment growth was altered by blue or red light instead of auxin in the presence or absence of mechanical stress. It is concluded from these results that growth changes, elicited by auxin, light, etc., and mechanical stress affect microtubule orientation through a common signal perception and transduction chain.Abbreviations IAA indole-3-acetic acid (auxin) - MT cortical microtubule     

IgG rheumatoid factors isolated by the surface-displaying phage library technique

 Our analysis of IgG rheumatoid factors (RFs) from three patients with rheumatoid arthritis (RA) revealed that most contained significant numbers of skewed mutations per V region, suggesting that these RFs arose from antigen-driven responses. To further study IgG RFs in RA, we used pComb3 vector to construct an IgG1,λ combinatorial antibody library from a synovial fluid sample. After panning against human IgG, Fab fragments from 71/96 phage clones bound to Fc-coated wells. Sequence analysis of 20 randomly chosen Fc-binders showed that 17 (85%) clones had identical heavy (H) and light (L) chain V regions, represented by Humha311 and Humla211, respectively. Of the remaining three clones, two had the same Humla211 L chain, but each with a different H chain V region. All the putative germline V genes for these RFs also encode RF in RA patients. However, none of these RF V regions are similar to those of the two IgG RFs derived by the hybridoma technique from the same synovial fluid. The Humha311 H chain has two frameshifts: a one-base insertion upstream of the JH region and a four-base deletion near the end of the CH1 region, resulting in a mainly unconventional amino acid sequence in the CH1 region. In the future, it will be important to study the presence of IgG molecules with such unconventional CH1 amino acid sequences, and the effects of these amino acid sequences on the structures and immunological properties of the IgG molecules. Received: 4 September 1996 / Revised: 22 October 1996     

Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs.     

Simultaneous rapid high-performance liquid chromatographic determination of phenytoin and its prodrug, fosphenytoin in human plasma and ultrafiltrate

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of phenytoin and fosphenytoin, a prodrug for phenytoin, in human plasma and plasma ultrafiltrate is described. For plasma, the method involves simple extraction of drugs with diethyl ether and evaporation of solvent, followed by injection of the reconstituted sample onto a reversed-phase C₁₈ column. Plasma ultrafiltrate is injected directly into the HPLC column. Compounds are eluted using an ion-pair mobile phase containing 20% acetonitrile. The eluent is monitored by UV absorbance at 210 nm. The fosphenytoin standard curves are linear in the concentration range 0.4 to 400 μg/ml for plasma and 0.03 to 80 μg/ml for ultrafiltrate. Phenytoin standard curves are linear from 0.08 to 40 μg/ml for plasma and from 0.02 to 5.0 μg/ml for ultrafiltrate. No interferences with the assay procedure were found in drug-free blank plasma or plasma ultrafiltrate. Relative standard deviation for replicate plasma or ultrafiltrate samples was less than 5% at concentrations above the limit of quantitation for both within- and between-run calculations.     

Phylogenetic Relationships within Paxillus s. I. (Basidiomycetes, Boletales): Separation of a Southern Hemisphere Genus

Abstract: This study aimed to test the monophyly and present a generic concept of Paxillus s. I. Of special interest were the Southern hemisphere representatives of this genus. Using the primer combination LR0R-LR5, DNA sequence data were generated from the nuclear encoded 28S gene. A data set containing 900 bases was created for 30 different species. Phylogenetic analysis showed 3 main monophyletic groups, also supported by morphological, anatomical, ecological and chemical data. Taxonomic interpretation of these clearly differentiated groups corresponded to the genera PaxilIus s. str. and Tapinella and lead to the proposal of a new Southern hemisphere genus, Austro-paxillus , mycorrhizal with Nothofagus . New combinations are proposed.