High purity preparations of higher plant vacuolar H+-ATPase reveal additional subunits. Revised subunit composition

A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20-25 mumol/mg.min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67- and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase. The ATPase of tonoplast vesicles is subject to MgATP-dependent cold inactivation, and the conditions for inactivation are identical to those for the bovine chromaffin granule H+-ATPase (Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 3577-3582). Cold inactivation is accompanied by the detachment of five major polypeptides of 67, 55, 52, 44, and 32 kDa from the membrane, and all five components co-migrate with the corresponding polypeptides of the purified ATPase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 100- and 16-kDa polypeptides of the ATPase are not removed from the membrane during cold inactivation, but the latter can be purified to homogeneity by chloroform:methanol extraction of the fast protein liquid chromatography-purified enzyme. It is concluded that the tonoplast H+-ATPase is constituted of 6-7 major polypeptides organized into a peripheral sector comprising the 67-, 55-, 52-, 44-, and 32-kDa components and an integral sector consisting of the 100- and 16-kDa polypeptides. The V-type H+-ATPase from animal endomembranes and higher plant vacuolar membranes therefore have remarkably similar subunit compositions and gross topographies.     

Predicting Carriers of Ongoing Selective Sweeps without Knowledge of the Favored Allele

Methods for detecting the genomic signatures of natural selection have been heavily studied, and they have been successful in identifying many selective sweeps. For most of these sweeps, the favored allele remains unknown, making it difficult to distinguish carriers of the sweep from non-carriers. In an ongoing selective sweep, carriers of the favored allele are likely to contain a future most recent common ancestor. Therefore, identifying them may prove useful in predicting the evolutionary trajectory—for example, in contexts involving drug-resistant pathogen strains or cancer subclones. The main contribution of this paper is the development and analysis of a new statistic, the Haplotype Allele Frequency (HAF) score. The HAF score, assigned to individual haplotypes in a sample, naturally captures many of the properties shared by haplotypes carrying a favored allele. We provide a theoretical framework for computing expected HAF scores under different evolutionary scenarios, and we validate the theoretical predictions with simulations. As an application of HAF score computations, we develop an algorithm (PreCIOSS: Predicting Carriers of Ongoing Selective Sweeps) to identify carriers of the favored allele in selective sweeps, and we demonstrate its power on simulations of both hard and soft sweeps, as well as on data from well-known sweeps in human populations.     

Nutritional Aspects of Dissimilatory Sulfate Reduction in the Human Large Intestine

In contrast to other anaerobic ecosystems, such as marine and estuarine sediments, there is a lack of information on the nutritional requirements of human gut sulfate-reducing bacteria (SRB). Various substrates stimulated sulfate reduction in mixed culture, including short-chain fatty acids and other organic acids, alcohols, and amino acids (but not sugars or aromatic compounds). However, the use of sodium molybdate as a specific inhibitor of sulfate reduction caused an accumulation of ethanol and malonate only, and reduced the rate of utilization of lactate. This indicates the importance of these electron donors for sulfate reduction. Since ethanol and lactate are primarily utilized by members of the Desulfovibrio genus, the results suggest a physiologically important role for this group.  Experiments with two strains of Desulfovibrio desulfuricans isolated from human feces demonstrated that both were able to reduce sulfite, thiosulfate or nitrate in the absence of sulfate. In addition, one strain (DsvUC1) was able to grow by fermentative metabolism, although the second strain (DsvFD1) showed more restricted fermentative growth. The data indicate that desulfovibrios are ecologically the most significant group of SRB in the human colon, and that colonic isolates belonging to this genus are versatile, in terms of both the electron acceptors and donors that they are able to utilize. Received: 24 March 1997 / Accepted: 10 June 1997