On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions

Using lacl-Z fusion strains of Escherichia coli we have devised systems that detect deletions of varying lengths. We examined deletions 700-1000 base pairs long, and genetically characterized over 250 spontaneous deletions. Of these, we analyzed 24 by direct DNA sequencing and 18 by inspection of restriction fragment patterns. Deletions of this size occur almost exclusively at short repeated sequences in both (recA+ and recA- strain backgrounds, but are detected 25-fold more frequently in a recA+ background. The frequency of deletion formation correlates with the extent of homology between the short repeated sequences, although other factors may be involved. The largest hotspot, which accounts for 60% of the deletions detected, involves the largest homology in the system (14 of 17 base pairs). Altering a single base pair within this homology reduces deletion incidence by an order of magnitude. We discuss possible mechanisms of deletion formation and consider its relationship to the excision of transposable elements.     

Termination and activation of store‐operated cyclic AMP production

Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1‐AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca²⁺] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this ‘store‐operated’ pathway requires the ER Ca²⁺ sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET‐based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store‐operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca²⁺ entry via the plasma membrane Ca²⁺ channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca²⁺/cAMP crosstalk system.     

The epidemiologic transition in a frontier town — Manti,Utah: 1849–1977

Census reports and information in burial records of Manti, Utah from 1849 to July 1977 are examined in order to (1)document mortality trends and differentials by age, sex, cause-of-death, and seasonality as Manti passed from a frontier settlement to a rural agricultural community; and (2) ascertain whether the shifts in the cause-of-death structure follow those patterns outlined by Omran (1971, 1974, 1977) in his theory of the epidemiologic transition. Findings parallel patterns suggested by Omran. Major factors accounting for mortality reductions are (1) elimination of the population's dependence upon a contaminated water supply, and (2) adoption of medical advances as they became available.     

Identification of the phosphorylated sites of phosphofructokinase from skeletal muscle after in vivo and in vitro phosphorylation.

Phosphofructokinase from mice muscle was radioactively labelled either in vivo by the injection of [³²P]-phosphate or in vitro by the incubation with cAMP-dependent protein kinase and [γ-³²P]-ATP. Two labelled peptides were obtained after tryptic digestion in either case showing that at least two sites were phosphorylated. Independent of the labelling method, the labelled peptides showed an analogous pattern on the peptide maps, indicating that both methods led to the phosphorylation of the same sites.     

The in vivo reproductive potential of density separated cells

Murine ascites cells (L1210, L5178Y, Ehrlich ascites) were labelled with ¹³¹I-iododeoxyuridine and subjected to buoyant density centrifugation on a continuous, linear Ficoll gradient. Cell losses sustained during density centrifugation were evaluated by recording the amount of ¹³¹I recovered in the final cell fractions. The viability and proliferative capacity of the density separated cells were tested by monitoring the rate of ¹³¹I excretion following inoculation of the recovered cells into new, non-radioactive hosts.Density separation in Ficoll appeared to cause few, if any, adverse effects. Cell recovery under properly regulated experimental conditions was virtually complete (97% or higher). The reproductive potential of density-separated cells was identical to that of control cells. However, considerable cell mortality could be induced by permitting cellular aggregation in medium free of antiagglutinin or by exposure of excessive quantities of cells to a density gradient.Viability indices obtained with trypan blue proved unsuitable for predicting long-term survival. In some experiments the trypan blue data provided a 90–100% viability reading when in fact the entire cell population had been inactivated by irradiation or heat incubation. Since the trypan blue test also did not reveal the full extent of mortality among aggregated cells or cells recovered from overloaded gradients, it was concluded that the dye exclusion test, in spite of its utility for monitoring immediate cell death and membrane destruction, was of limited value for evaluating the reproductive potential of mammalian cells.