Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage

Summary Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD) that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at 180 copies per uninduced cell and was measured at 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.     

Carotenoids and abscisic acid (ABA) biosynthesis in higher plants

Recent research has revealed that abscisic acid (ABA), synthesised in response to water stress, is an apo-carotenoid. Two potential carotenoid precursors, 9'- cis -neoxanthin and 9- cis -violaxanthin, have been identified in light-grown and etiolated leaves, and in roots of a variety of species. Experiments utilizing etiolated Phaseolus vulgaris leaves and deuterium oxide strongly suggest that 9'- cis -neoxanthin, synthesised from all- trans -violaxanthin, is the immediate pre-cleavage precursor of ABA. The cleavage of 9'- cis -neoxanthin, performed by an inducible and specific dioxygenase, is likely to be the rate-limiting step in ABA biosynthesis. Any apocarotenoids formed as by-products of cleavage are probably rapidly degraded by lipoxygenase or related enzymes. After cleavage xanthoxin is converted via ABA-aldehyde to ABA by constitutive enzymes in the cytosol.     

Study of glucose starvation in excised maize root tips

Excised maize (Zea mays) root tips were used to follow the effects of a prolonged glucose starvation. Respiration rate began to decrease immediately after excision, reaching 30 to 40% of its initial value after 20 hours, and then declined more slowly until death of the tissues, which occurred after 200 hours of starvation. During the whole process, respiration could be uncoupled by 2,4-dinitrophenol and the energy charge remained high. These results suggest that in excised maize root tips, respiration rate is essentially limited by the rate of biosyntheses (ATP-utilizing processes) rather than mitochondrial number. During starvation the sugar content sharply decreased for the first 20 hours and reached zero at 120 hours. Following root excision, proteins and lipids were continuously degraded and were virtually the only substrates for respiration and biosyntheses after 20 hours of starvation. Over the first 90 hours of starvation, enzymic activities related to sugar metabolic pathways and the Krebs cycle decreased to 20% or less of their initial activity. Starvation was reversible only for the first 80 to 90 hours. Between 80 and 100 hours, there was a sharp fall in intracellular osmolarity and a 25% loss in the dry weight. The irreversibility may be due, as in senescence, to a change in membrane selective permeability.     

The trophic-dynamic aspect of ecology

Recent progress in the study of aquatic food-cycle relationships invites a reappraisal of certain ecological tenets. Quantitative productivity data provide a basis for enunciating certain trophic principles, which, when applied to a series of successional stages, shed new light on the dynamics of ecological succession. From “The trophic-dynamic aspect of ecology” by R. L. Lindeman.Ecology, Vol. 23, pp. 399–418 (1942).     

Restoration and creation of freshwater wetlands using seed banks

The minimum information about a seed bank needed for a wetland restoration or creation project is a species list. There are two basic techniques for determining the composition of seed banks: (1) mechanical separation of seeds from a volume of soil and (2) germination of seeds from a volume of soil under appropriate environmental conditions. The latter method always gives biased results. It is best to collect as many random samples as possible when sampling a wetland seed bank. These can be combined as needed for processing. Field studies in India have demonstrated that vestigial seed banks can be used to re-establish a former vegetation type in a monsoonal wet-land that had become overgrown by a species of grass. In less than a year, 9 of 1 I species in the vestigial seed bank were found growing in areas cleared of the grass. Vestigial seed banks of drained prairie wetlands in the northcentral United States contained a few wetland species after 70 years, although species diversity and seed density declined significantly after 20 to 30 years of drainage and cultivation. In Florida, U.S.A., wetlands have been established in strip-mined areas using donor soils from existing wetlands. Newly established wetlands quickly developed a dense cover of vegetation, although this vegetation often lacked many desirable wetland species. Experimental studies of soil moisture conditions using a seed bank from the Delta Marsh, Canada, demonstrated that soil moisture affected both the total number of seeds, and the relative proportion of seeds of each species that germinated from a seed bank. The density of seedlings of emergent wetland species in the treatments was directly proportional to soil moisture, while that of terrestrial annuals was inversely proportional. Emergent species made up nearly 90% of the seedlings in the wettest treatment and 0% in the driest.From a paper presented at the Third International Wetlands Conference, 19–23 September, 1988, University of Rennes, France.     

Mouse Chromosome 14

Chair of Committee for Mouse Chromosome 14