The role of a conserved dodecamer sequence in yeast mitochondrial gene expression

All mRNAs on the yeast mitochondrial genome terminate at a conserved dodecamer sequence 5'-AAUAAUAUUCUU-3'. We have characterized two mutants with altered dodecamers. One contains a deletion of the dodecamer at the end of the var1 gene, and the other contains two adjacent transversions in the dodecamer at the end of the reading frame of fit1, a gene within the omega+ allele of the 21S rRNA gene. In each mutant, expression of the respective gene is blocked completely. A dominant nuclear suppressor, SUV3-1, was isolated that suppresses the var1 deletion but is without effect on the fit1 dodecamer mutations. Unexpectedly, however, we found that SUV3-1 blocks expression of the wild-type fit1 allele by blocking processing at its dodecamer. SUV3-1 has pleiotropic effects on mitochondrial gene expression, affecting RNA processing, RNA stability, and translation. Our results suggest that RNA metabolism and translation may be part of a multicomponent complex within mitochondria.     

Studies on the Effect of Insulin-Like Growth Factor-I on Catecholamine Secretion from Chromaffin Cells

Chromaffin cells cultured in serum-free medium secreted a smaller percentage of their catecholamine stores in response to stimulation by high K+ (55 mM) than did cells cultured in serum-containing medium. Addition of insulin-like growth factor-I (IGF-I) to serum-free medium restored high K(+)-stimulated catecholamine secretion to the levels seen in serum-treated cultures. In contrast, addition of IGF-I to serum-containing medium had little effect on catecholamine secretion. These results suggest that serum contains IGF-I or another factor that maintains the secretory responsiveness of chromaffin cells. IGF-I not only enhanced high K(+)-stimulated catecholamine secretion, but also augmented secretion elicited by the nicotinic agonist dimethyl-phenylpiperazinium, the dihydropyridine agonist Bay K 8644, and Ba2+. IGF-I did not affect the dependence of catecholamine secretion on extracellular Ca2+ concentration nor did it affect the time course of secretion. Experiments using 45Ca2+ demonstrated that IGF-I treatment enhanced Ca2+ uptake into the cells. When cells were permeabilized by treatment with digitonin, Ca2(+)-dependent catecholamine secretion was slightly, but consistently, greater from IGF-I-treated cells than from untreated cells. Our results suggest that IGF-I may enhance catecholamine secretion partly by increasing Ca2+ entry into the cells and partly by affecting a step distal to Ca2+ entry.     

Endocytosis in the rat retinal pigment epithelium

Endocytosis in the retinal pigment epithelium (RPE) of rats was studied using horseradish peroxidase, microperoxidase and ferritin tracers. Tracer uptake was mediated by coated pits and coated vesicles. Coated pits formed at two discrete regions at the RPE plasma membrane: that portion of basal membrane directly opposing Bruch's membrane, and at the bases of the apical lamellae and villi. Two populations of coated vesicles were identified and distinguished by size, location and function. Large coated vesicles (91.8 +/- 14.7 nm in diameter) were located near the cell surface and incorporated tracer. Small coated vesicles (64.5 +/- 15.7 nm diameter) located more deeply within the cell were not tracer-labeled, and were often fused with the endoplasmic reticulum or the Golgi apparatus. Observations of the endocytic pathway in rat RPE cells are presented. Tracer was also found in organelles of the lysosomal system, e.g. the multivesicular body, but was not identified in the smooth endoplasmic reticulum or Golgi apparatus.     

Conformational changes in the alpha- and beta-subunits of the insulin receptor identified by anti-peptide antibodies

The structure of the insulin receptor was studied with polyclonal antibodies obtained from rabbits which were immunized with synthetic peptides having a sequence identity to three regions of the alpha-subunit and five regions of the beta-subunit. None of the alpha-subunit antibodies including alpha-Pep8 (residues 40-49 (Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzzelli, L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O.M., and Ramachandran, J. (1985) Nature 313, 756-761), alpha-Pep7 (12 amino acid C-terminal extension (Ebina, Y., Ellis, L., Jarnagin, K., Ederly, M., Graf, L., Clauser, E., Ou, J.-H., Masiar, F., Kan, Y.W., Goldfine, I.D., Roth, R.A., and Rutter, W.J. (1985) Cell 313, 747-758], or alpha-Pep6 (residues 1-7, 9) immunoprecipitated the human insulin receptor solubilized from IM-9 lymphocytes; however, alpha-Pep8 immunoprecipitated the dithiothreitol-reduced receptor. Antibodies prepared against the N terminus of the beta-subunit (alpha-Pep5, residues 780-790) and the ATP binding site (alpha-Pep3, residues 1013-1022) did not react with the intact receptor under any conditions; however, antibodies to the C terminus of the beta-subunit (alpha-Pep1, residues 1314-1324) and to the juxta-membrane region (alpha-Pep3, residues 952-962) immunoprecipitated the solubilized receptor in both its phosphorylated and nonphosphorylated forms. In contrast, the antibody reactive with the regulatory region of the beta-subunit which contains the major autophosphorylation sites (alpha-Pep2, residues 1143-1154) only precipitated the phosphorylated form. Thus the conformation of the extracellular domain of the receptor is rigid and stabilized by disulfide bonds, whereas several regions of the intracellular domain are accessible to antibodies and undergo conformational changes during autophosphorylation.     

Length changes in the joining segment between domains 5 and 6 of a group II intron inhibit self-splicing and alter 3' splice site selection.

Domain 5 (D5) and domain 6 (D6) are adjacent folded hairpin substructures of self-splicing group II introns that appear to interact within the active ribozyme. Here we describe the effects of changing the length of the 3-nucleotide segment joining D5 to D6 [called J(56)3] on the splicing reactions of intron 5 gamma of the COXI gene of yeast mitochondrial DNA. Shortened variants J(56)0 and J(56)1 were defective in vitro for branching, and the second splicing step was performed inefficiently and inaccurately. The lengthened variant J(56)5 had a milder defect-splicing occurred at a reduced rate but with correct branching and a mostly accurate 3' splice junction choice. Yeast mitochondria were transformed with the J(56)5 allele, and the resulting yeast strain was respiration deficient because of ineffective aI5 gamma splicing. Respiration-competent revertants were recovered, and in one type a single joiner nucleotide was deleted while in the other type a nucleotide of D6 was deleted. Although these revertants still showed partial splicing blocks in vivo and in vitro, including a substantial defect in the second step of splicing, both spliced accurately in vivo. These results establish that a 3-nucleotide J(56) is optimal for this intron, especially for the accuracy of 3' splice junction selection, and indicate that D5 and D6 are probably not coaxially stacked.     

Factors influencing plasma zinc levels in low-income pregnant women

Plasma zinc (Zn) concentrations were measured in 4376 indigent women (86% African-American), at a mean (±SD) gestational age of 15 (±7.8) wk to determine the relationship between various maternal characteristics and plasma Zn levels during pregnancy. Mean plasma Zn levels were lower in African-American women than in Caucasian women, in multiparous women than in primiparous women, and in women with body weight >69.9 kg than in those with body weight ≤69.9 kg (p≤0.001 for each comparison). There were no significant differences related to maternal age, marital status, education, or smoking habit. Multiple regression analysis, including maternal prepregnancy weight, race, age, parity, smoking habit, education, and marital status indicated that race, parity, and pregnancy weight were significantly associated with maternal plasma Zn levels, adjusted for gestational age. Maternal race was the best predictor of plasma Zn concentrations among the population of pregnant women studied A significant proportion of variance in maternal plasma Zn levels remained unexplained after taking into account various maternal characteristics. The reasons for lower plasma Zn levels in African-American women, compared to Caucasian women, during pregnancy are unknown.     

Purified moniliformin does not affect the force or rate of contraction of isolated guinea pig atria

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F₂ generation. The heritability estimates of crosses GFA-2 × NC-V11 and Tifton-8 × NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.Cooperative investigation of the USDA-ARS and the University of Georgia, College of Agriculture.     

Measures of geographic range size: the effects of sample size

A number of methods have been used for quantifying the sizes of the geographic ranges of species. The consequences of different levels of sampling (the proportion of actual spatial occurrences) are explored for eight of these, using data on the occurrences of butterfly species on a 10 × 10 km grid across Britain. For all methods, the percentage error of estimation (PEE) decreases with the number of 10 × 10 km squares which a species occupies, most rapidly for extent measures, and more rapidly for area measures than for measures of numbers of units occupied. The rate of decline in PEE itself falls as sampling effort increases. At a given sampling level, rank correlations between range sizes measured by different methods are generally high, but there is no consistent change in the magnitude of these correlations as the level of sampling increases. The composition of the set of species with the smallest range sizes changes with the level of sampling.     

A UV-induced, Mg(2+)-dependent crosslink traps an active form of domain 3 of a self-splicing group II intron.

In vitro irradiation of a15 gamma group II intron RNA with low doses of 254 nm UV light induces a single major crosslink. This crosslink was mapped within the domain 3 substructure of this RNA and one of the participating nucleotides was identified. When an RNA containing only the domain 3 substructure is irradiated under the same conditions, an intramolecular crosslink forms between two specific pyrimidines, one of them identical to the nucleotide crosslinked in the full-length intron RNA. In both RNAs, the crosslink is magnesium ion-dependent and photoreversible. A trans assay for domain 3 function was developed and used to find that the crosslinked domain 3 RNA remains highly reactive. This suggests that crosslinking has trapped a functional, Mg(2+)-induced folded state of this group II intron substructure and that this folding is probably independent of the other domains of the intron.