Identification of the bud emergence gene BEM4 and its interactions with rho-type GTPases in Saccharomyces cerevisiae.

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators

Controller Design for the ST7 Disturbance Reduction System

The Space Technology 7 experiment will perform an on-orbit system-level validation of two specific Disturbance Reduction System technologies: a gravitational reference sensor employing a free-floating test mass and a set of micro-Newton colloidal thrusters. The Disturbance Reduction System is designed to maintain a spacecraft's position with respect to the free-floating test mass to less than 10 nm/ square root of Hz, over the frequency range 10(exp -3) Hz to 10(exp -2) Hz. This paper presents the design and analysis of the coupled drag-free and attitude control system that closes the loop between the gravitational reference sensor and the micro-Newton thrusters while incorporating star tracker data at low frequencies. The effects of actuation and measurement noise and disturbances on the spacecraft and test masses are evaluated in a seven-degree-of-freedom planar model incorporating two translational and one rotational degrees of freedom for the spacecraft and two translational degrees of freedom for each test mass

Heterogeneity of alpha-cardiac myosin heavy chains in a small marsupial, Antechinus flavipes, and the effect of hypothyroidism on its ventricular myosins

The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin components were found: V1a, V1b, V1c, V2 and V3, in order of decreasing mobility. In western blots, V1a, V1b, and V1c reacted with anti-a-MyHC antibody, but not with anti-B-MyHC, whereas V2 and V3 reacted with anti-B-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which bound anti-a-MyHC antibody while the third bound anti-B-MyHC antibody. We conclude that V1a, V1b, V1c are triplets arising from the dimerization of two distinct a-MyHC isoforms. Hypothyroidism, veriWed by metabolic studies, decreased a-MyHC content significantly (t-test, P < 0.001) from 91.6 5.9% (SEM, n = 4) in control animals to 67.2 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in B-MyHC content. We conclude that in adult marsupials, ventricular myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians

The investigation of optimal bombardment parameters for transient and stable transgene expression in sorghum

© 2001 Society for In Vitro Biology The original publication can be found at www.springerlink.comThis report outlines the development of optimized particle inflow gun (PIG) parameters for producing transgenic sorghum (Sorghum bicolor (L.) Moench). Both transient and stable expression were examined when determining these parameters. The uidA reporter gene (GUS) encoding β-glucuronidase was used in transient experiments and the green fluorescent protein (GFP) used to monitor stable expression. Initially, optimization was conducted using leaf segments, as the generation of sorghum callus in sufficiently large quantities is time-consuming. Following leaf optimization, experiments were conducted using callus, identifying a high similarity between the two tissue types (r s=0.83). High levels of GUS expression were observed in both leaf and callus material when most distant from the DNA expulsion point, and using a pressure greater than 1800 kPa. A higher level of expression was also observed when the aperture of the helium inlet valve was constricted. Using the optimized conditions (pressure of 2200 kPa, distance to target tissue of 15 cm from the expulsion point, and the aperture of the helium inlet valve at one full turn), three promoters (Ubiquitin, Actinl and CaMV 35S) were evaluated over a 72-h period using GUS as the reporter gene. A significantly higher number of GUS foci were counted with the Ubiquitin construct over this period, compared to the Actinl and CaMV 35S constructs. Stable callus sectors (on 2 mg 1−1 bialaphos) with GFP expression were visualized for as long as 6 wk post-bombardment. Using this optimized protocol, several plants were regenerated after having been bombarded with the pAHC20 construct (containing the bar gene), with molecular evidence confirming integration.Jason A. Able, Carl Rathus and Ian D. Godwi