Studies of the mechanism of spontaneous germline ecotropic provirus acquisition in mice.

SWR/J--RF/J hybrid mice spontaneously acquire new germline ecotropic proviruses at high frequency. We have performed ovarian transplantation and in situ hybridization studies to delineate the mechanism and developmental stage of germline provirus acquisition. In addition, we have developed a novel, efficient and simple method to introduce single copy proviruses into the mouse germline. The results reported here have direct implications for understanding how proviruses are acquired in the germline, for using murine leukemia viruses as insertional mutagens, and for using retroviral vectors to introduce foreign genes into the mouse germline.     

Cadmium inhibition of the erythrocyte Ca2+ pump. A molecular interpretation

The effects of cadmium (Cd2+) on transmembrane Ca2+ transport and on the membrane permeability for Ca2+ were studied in human erythrocytes. The erythrocyte Ca2+ pump is inhibited competitively by Cd2+ via interaction with the Ca2+ transport site of the carrier and not via interaction with its activator calmodulin. The affinity of the Ca2+ pump for Cd2+ is extremely high (KI = 2.0 nM Cd2+). Cd2+ (less than or equal to 10(-4) M) does not alter the membrane permeability for Ca2+. We conclude that the pivotal mechanism in the toxic action of Cd2+ is the inhibition of Ca2+-ATPase mediated Ca2+ extrusion. As a result Cd2+ disturbs intracellular Ca2+ homeostasis and may increase cytosolic Ca2+ (Ca2+i) to toxic levels.     

Dynamics of IS-related genetic rearrangements in resting Escherichia coli K-12

An analysis of restriction fragment length polymorphism (RFLP) using eight residential insertion sequence (IS) elements as hybridization probes reveals that the genome of resting bacteria is more dynamic than it was long believed. Escherichia coli strains stored in agar stabs for up to 30 yr accumulate a genetic variation which is correlated to time of storage. This spontaneous mutagenesis is often IS-specific, with particularly high activity for IS5, and thus suggests that transpositional DNA rearrangements are a major cause for the observed genetic polymorphism. The RFLP patterns indicate a burst of IS30 transposition to occur occasionally. Mutation rate is estimated by two different methods to roughly 10(-5) IS-related DNA rearrangements per bacterial chromosome per hour of storage for the eight IS elements studied. A pedigree derived from the RFLP data reveals that populations had evolved independently in each stab and showed no signs of convergence. Relics of an assumed ancestral population were still present in the stab cultures, but the elder stabs provided mostly mutants. These results indicate that cells placed under nutritional deprivation might have a highly plastic genome and suggest that such plasticity might play an adaptive role.      

Three photoconvertible forms of green fluorescent protein identified by spectral hole-burning.

Several studies have led to the conclusion that, in the green fluorescent protein (GFP) of the jellyfish Aequorea victoria, a photoconversion involving excited-state proton transfer occurs from an A- to a B-form, while an intermediate I-form was held responsible for the green fluorescence. Here we have identified the I-form of wild-type GFP in absorption, located the 0-0 transitions of all three forms A, B and I, and determined vibrational frequencies of the ground and excited states. The intrinsically narrow 0-0 transitions are revealed by the wavelengths at which holes can be burnt. The pathways of photointerconversion are unraveled by excitation, emission and hole-burning spectroscopy. We present an energy-level scheme that has significant implications for GFP-mutants, which likewise can occur in the three photo-interconvertible forms.