Genetic suppression of intronic +1G mutations by compensatory U1 snRNA changes in Caenorhabditis elegans

Mutations to the canonical +1G of introns, which are commonly found in many human inherited disease alleles, invariably result in aberrant splicing. Here we report genetic findings in C. elegans that aberrant splicing due to +1G mutations can be suppressed by U1 snRNA mutations. An intronic +1G-to-U mutation, e936, in the C. elegans unc-73 gene causes aberrant splicing and loss of gene function. We previously showed that mutation of the sup-39 gene promotes splicing at the mutant splice donor in e936 mutants. We demonstrate here that sup-39 is a U1 snRNA gene; suppressor mutations in sup-39 are compensatory substitutions in the 5' end, which enhance recognition of the mutant splice donor. sup-6(st19) is an allele-specific suppressor of unc-13(e309), which contains an intronic +1G-to-A transition. The e309 mutation activates a cryptic splice site, and sup-6(st19) restores splicing to the mutant splice donor. sup-6 also encodes a U1 snRNA and the mutant contains a compensatory substitution at its 5' end. This is the first demonstration that U1 snRNAs can act to suppress the effects of mutations to the invariant +1G of introns. These findings are suggestive of a potential treatment of certain alleles of inherited human genetic diseases.     

Estimates of extreme sperm production: morphological and experimental evidence from reproductively promiscuous fairy-wrens (Malurus)

When females mate multiply, the competition among males for fertilizations occurs cryptically within the reproductive tract of the female. Under such sperm competition, the amount of spermatogenic tissue, the efficiency of sperm production and the capacity for sperm storage should all be under strong selection. Males of several species of fairy-wrens (Malurus) have been found to store large numbers of sperm for ejaculation, a suggested adaptation to male-biased sex ratios and high levels of reproductive promiscuity. In this study we examined the histology of testes of three species of fairy-wren: the splendid, Malurus splendens melanotus, variegated, M. lamberti assimilis, and white-winged fairy-wren, M. leucopterus leuconotus, as well as the quantity and quality (viability) of sperm in captive male splendid fairy-wrens to estimate the temporal pattern of sperm production and depletion. The daily rates of sperm production in all three species were high: for splendid, for variegated and for white-winged fairy-wrens, and the efficiency of sperm production (sperm per day per g of body mass) was several orders of magnitude greater than values reported for other domestic and wild species. For splendid fairy-wrens, the estimates of sperm production varied with the method used for estimation (range of estimates ). Splendid fairy-wren males held in captivity replenished their sperm reserves within 12 h, which is faster than that reported for most other species thus far. There was a negative correlation between length of sampling interval and the proportion of live sperm in ejaculate samples, indicating that males that copulate more frequently have higher-quality ejaculates. Together, both experimental and morphological estimates suggest that all three species of fairy-wren invest considerable energy in the production as well as the storage of massive numbers of sperm. Intra- and interspecific variation in the rates of sperm production is most likely linked to differences in the operational sex ratios of social groups and the opportunity for extragroup parentage.     

A method for measuring disulfide reduction by cultured mammalian cells: relative contributions of glutathione-dependent and glutathione-independent mechanisms

A method is described for measuring bioreduction of hydroxyethyl disulfide (HEDS) or alpha-lipoate by human A549 lung, MCF7 mammary, and DU145 prostate carcinomas as well as rodent tumor cells in vitro. Reduction of HEDS or alpha-lipoate was measured by removing aliquots of the glucose-containing media and measuring the reduced thiol with DTNB (Ellman's reagent). Addition of DTNB to cells followed by disulfide addition directly measures the formation of newly reduced thiol. A549 cells exhibit the highest capacity to reduce alpha-lipoate, while Q7 rat hepatoma cells show the highest rate of HEDS reduction. Millimolar quantities of reduced thiol are produced for both substrates. Oxidized dithiothreitol and cystamine were reduced to a lesser degree. DTNB, glutathione disulfide, and cystine were only marginally reduced by the cell cultures. Glucose-6-phosphate deficient CHO cells (E89) do not reduce alpha-lipoate and reduce HEDS at a much slower rate compared to wild-type CHO-K1 cells. Depletion of glutathione prevents the reduction of HEDS. The depletion of glutathione inhibited reduction of alpha-lipoate by 25% and HEDS by 50% in A549 cells, while GSH depletion did not inhibit alpha-lipoate reduction in Q7 cells but completely blocked HEDS reduction. These data suggest that the relative participation of the thioltransferase (glutaredoxin) and thioredoxin systems in overall cellular disulfide reduction is cell line specific. The effects of various inhibitors of the thiol-disulfide oxidoreductase enzymes (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), arsenite, and phenylarsine oxide) support this conclusion.     

Mitochondrial DNA-depleted neuroblastoma (Rho degrees) cells exhibit altered calcium signaling

To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho degrees ). Basal cytosolic calcium levels were slightly but significantly reduced in Rho degrees cells. The impact of chronic depletion of mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium transients generated in Rho degrees cells following application of carbachol were more rapid than those in SH-SY5Y cells. A plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho degrees cells. The rapid calcium transients in Rho degrees cells were due, in part, to increased reliance on Na(+)/Ca(2+) exchange activity at the plasma membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular calcium stores. Rho degrees cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen in Rho degrees cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a consequence of chronic depletion of mitochondrial DNA.     

Glucose-6-phosphate dehydrogenase and the oxidative pentose phosphate cycle protect cells against apoptosis induced by low doses of ionizing radiation

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP(+). Hence, under normal growth conditions, where NADPH levels exceed NADP(+) levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD(-)) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD(-) cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD(-) cells exhibited a significant enhancement in the alpha component of radiation-induced cell killing, while the values obtained for the beta component were similar in both the G6PD(-) and wild-type CHO cell lines. Here we report that the enhanced alpha component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD(-) cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD(-) cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD(-) cell line     

Characterization of plasmid pRT1 from Pyrococcus sp. strain JT1

We discovered a 3,373-bp plasmid (pRT1) in the hyperthermophilic archaeon Pyrococcus sp. strain JT1. Two major open reading frames were identified, and analysis of the sequence revealed some resemblance to motifs typically found in plasmids that replicate via a rolling-circle mechanism. The presence of single-stranded DNA replication intermediates of pRT1 was detected, confirming this mode of replication.     

Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex

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Distribution and oxidation of malondialdehyde in mice

The metabolism of melondialdehyde (MDA) by male and female Swiss mice was investigated. Distribution of an i.p. dose of MDA is rapid and uniform throughout the body. Conversion of ¹⁴C-labeled MDA to CO₂ is complete 4 hours after an i.p. dose of 5 μmol to 200 μmol with no signs of short term toxicity. The yields of CO₂ from [1-¹⁴C]-β-alanine, [3-¹⁴C]-β-alanine, [1-¹⁴C]-sodium acetate, and [2-¹⁴C]-sodium acetate were also determined. Comparison of the yields of CO₂ from this series of compounds suggests the intermediacy of malonic semialdehyde in the metabolism of MDA. High doses (600 μmol) of β-alanine or acetate given prior to ¹⁴C-MDA reduced the yield of ¹⁴CO₂. Ethanol and disulfiram were both inhibitors of MDA metabolism, indicating the involvement of aldehyde dehydrogenase in the oxidation of MDA.These data demonstrate the ability of animal tissues to rapidly remove exogeneously administered MDA. They also have implications with respect to the possible pathological consequences of MDA generation.     

3H]acetylcholine synthesis in cultured ciliary ganglion neurons: effects of myotube membranes

Avian ciliary ganglion neurons in cell culture were examined for the capacity to synthesize acetylcholine (ACh) from the exogenously supplied precursor, choline. Relevant kinetic parameters of the ACh synthetic system in cultured neurons were found to be virtually the same as those of the ganglionic terminals in the intact iris. Neurons were cultured in the presence of and allowed to innervate pectoral muscle; this results in an capacity for ACh synthesis. In particular, the ability to increase ACh synthesis upon demand after stimulation is affected by interaction with the target. This effect is shown to be an acceleration of the maturation of the cultured neurons. Lysed and washed membrane remnants of the muscle target were able to duplicate, in part, this effect of live target tissue on neuronal transmitter metabolism. Culture medium conditioned by muscle, and by the membrane remnants of muscle, was without significant effect. Thus, substances secreted into the medium do not play a major role in this interaction. Neurons cultured with either muscle or muscle membrane remnants formed large, elongate structures on the target membrane surface. These were not seen in the absence of the target at the times examined. This morphological difference in terminal-like structures may parallel the developmental increases in size and vesicular content of ciliary ganglion nerve terminals in the chick iris, and may relate to the increased ACh synthetic activity. The results suggest that direct contact with an appropriate target membrane has a profound, retrograde influence upon neuronal metabolic and morphological maturation.