Catalytic activity of cytochrome oxidase and cytochrome c in apolar solvents containing phospholipids and low amounts of water

Cytochrome c and cytochrome oxidase, in bovine heart submitochondrial particles and in their purified forms, were transferred to a ternary system that contained phospholipids (10 mg/ml toluene), the apolar solvent toluene, and water at concentrations of 13-15 microliters (high water) and 3 microliters (low water) per milliliter of toluene. When the enzymes were transferred back to an all water system, they exhibited full catalytic capacity. In the low water ternary system, cytochrome c could be reduced by ascorbate introduced via inverted micelles. Also in this system, cytochrome oxidase was reduced by ascorbate and cytochrome c but its oxidation was highly impaired. Data on the kinetics of reduction by ascorbate of cytochrome c and cytochrome oxidase under these conditions are presented. Cytochrome oxidase reduced in the organic solvent by ascorbate failed to form a complex with CO, but formed a complex with cyanide introduced via inverted micelles. The oxidized and the ascorbate-reduced cytochrome oxidase-cyanide complex exhibited a trough at 415 nm and a peak at 433 nm. The extent and rate of formation of the cyanide complex were higher with the reduced form of cytochrome oxidase. To achieve protein-protein interactions (cytochrome c-cytochrome oxidase) in the ternary system, it was necessary to extract the two proteins together. There was no functional interaction when they were extracted separately and mixed. In the high water ternary system reduced cytochrome oxidase was not detected, and it oxidized ascorbate at a higher rate than in the low water system; however, this rate was several orders of magnitude lower than in aqueous media.     

A lacZ-pbpB gene fusion coding for an inducible hybrid protein that recognizes localized sites in the inner membrane of Escherichia coli.

An in-phase gene fusion consisting of the 5'-terminal 1,314 base pairs (bp) of the structural gene for beta-galactosidase (lacZ) and the 3'-terminal 1,644 bp of the structural gene coding for penicillin-binding protein 3 (pbpB) of Escherichia coli was constructed and cloned in the plasmid pDIAM64. The product of the fusion gene was a remarkably stable protein with an apparent molecular weight of 110,000 (p110) that retained the ability to covalently interact with beta-lactam antibiotics. The fusion protein was found associated with the membrane at low levels of induction, but it accumulated in the cytoplasm of cells induced for a long time as inclusion bodies of high density. Inclusion bodies were localized at defined positions corresponding to septal sites in all of the pDIAM64-containing strains tested except PAT84 and GD113 (which carry the ftsZ84 mutant allele). These findings indicate a possible role of the FtsZ protein in the integration of Pbp3 into the membrane and in septum localization during the cell division cycle.     

Production of pili on Vibrio parahaemolyticus

Electron microscopic examination showed that all strains of Vibrio parahaemolyticus examined had pili on their surface when the organism was grown on marine agar at 28 degrees C for 6-12 h. The pili were morphologically stable on heat treatment at 60 degrees C for 10 min, but both the lateral and polar flagella possessed by this organism were labile. No immunological cross-reactivity between pili of enterotoxigenic Escherichia coli and Vibrio cholerae non-01 and those of V. parahaemolyticus was observed.     

Thermostability of membrane systems in organic solvents

Two multisubunit enzymes of the inner mitochondrial membrane, cytochrome oxidase and the H+-ATPase may be transferred into highly apolar solvents as protein-lipid complexes. At 70 degrees C and an initial water concentration of 13 microliters per ml organic solvent (toluene), the half-life of the ATPase was approx. 11 h, whereas that of cytochrome oxidase was about 100 s. Thermostability of cytochrome oxidase could be increased more than 100-times by decreasing the water concentration to 3 microliters per ml toluene. At this latter concentration of water the half-life of the ATPase at 90, 80 and 70 degrees C was 5, 48 and 96 h, respectively.     

On the virtues and pitfalls of the molecular evolutionary clock

"Informational" macromolecules--i.e., proteins and nucleic acids--have in their sequences a register of evolutionary history. Zuckerkandl and Pauling suggested in 1965 that these molecules might provide a "molecular clock" of evolution. The molecular clock would time evolutionary events and make it possible to reconstruct phylogenetic history--the branching relationships among lineages leading to modern species. Kimura's neutrality theory postulates that rates of molecular evolution are stochastically constant and, hence, that there is a molecular clock. A variety of tests have shown that molecular evolution does not behave like a stochastic clock. The variance in evolutionary rates is much too large and thus inconsistent with the neutrality theory. This, however, does not invalidate the clock, but rather leaves it without a theoretical foundation to anticipate its properties. Sequence comparisons show that molecular evolution is sufficiently regular to serve in many situations as a clock, but uncertainty concerning the properties of the clock (for example, about the circumstances that may yield large oscillations in substitution rates from time to time or from lineage to lineage) demands that it be used with caution. Few DNA or protein sequences are known from organisms that range from closely related, e.g., different mammals, to very remote, e.g., mammals and fungi. One example is cytochrome c, which has an acceptable clockwise behavior over the whole span, in spite of some irregularities. Another example is the copper-zinc superoxide dismutase (SOD), which behaves like a very erratic clock. The SOD average rate of amino acid substitution per 100 residues per 100 million years (MY) is 5.5 when fungi and animals are compared, 9.1 when comparisons are made between insects and mammals, and 27.8 when mammals are compared with each other. The question is which mode is more common over broad evolutionary spans: the regularity of cytochrome c or the capriciousness of SOD? Additional data sets will be required in order to obtain the answer and to develop expectations about the accuracy of the clock in particular instances. Until such data exist, conclusions solely based on the molecular clock are potentially fraught with error.     

Fertility interactions in Drosophila: Theoretical model and experimental tests

The results of 11 experiments with Drosophila species show that fertility is not a reducible property: the fertility of a mating pair cannot be predicted from the average fertility of the two genotypes involved. We propose a model of fertility selection that does not assume additivity (or multiplicativity) but assumes random mating and that the genotypic frequencies are in Hardy-Weinberg equilibrium. Numerical simulations show that removal of the assumption of Hardy-Weinberg frequencies does not significantly change the equilibrium frequencies predicted by the model.     

Mitochondrial DNA evolution in theDrosophila nasuta subgroup of species

Summary TheDrosophila nasuta group consists of about 12 closely related species distributed throughout the Indo-Pacific region. They are of great interest because of their evolutionary idiosyncrasies including little morphological differentiation, the ability to intercross in the laboratory often producing fertile offspring, and substantial chromosomal evolution. Studies of metric traits, reproductive isolation, and chromosomal and enzyme polymorphisms have failed to resolve the phylogeny of the species. We report the results of a survey of the mitochondrial DNA (mtDNA) restriction patterns of the species. The phylogeny obtained is consistent with other available information and suggests thatD. albomicans may represent the ancestral lineage of the group. The amount of polymorphism in local populations (=1.0% per site) is within the typical range observed in other animals, includingDrosophila. The degree of differentiation between species is, however, low: the origin of the group is tentatively dated about 6–8 million years ago. This study confirms the usefulness of mtDNA restriction patterns for ascertaining the phylogeny of closely related species.     

Phylogeny of Drosophila and related genera inferred from the nucleotide sequence of the Cu,Zn Sod gene

The phylogeny and taxonomy of the drosophilids have been the subject of extensive investigations. Recently, Grimaldi (1990) has challenged some common conceptions, and several sets of molecular data have provided information not always compatible with other taxonomic knowledge or consistent with each other. We present the coding nucleotide sequence of the Cu,Zn superoxide dismutase gene (Sod) for 15 species, which include the medfly Ceratitis capitata (family Tephritidae), the genera Chymomyza and Zaprionus, and representatives of the subgenera Dorsilopha, Drosophila, Hirtodrosophila, Scaptodrosophila, and Sophophora. Phylogenetic analysis of the Sod sequences indicates that Scaptodrosophila and Chymomyza branched off the main lineage before the major Drosophila radiations. The presence of a second intron in Chymomyza and Scaptodrosophila (as well as in the medfly) confirms the early divergence of these two taxa. This second intron became deleted from the main lineage before the major Drosophila radiations. According to the Sod sequences, Sophophora (including the melanogaster, obscura, saltans, and willistoni species groups) is older than the subgenus Drosophila; a deep branch splits the willistoni and saltans groups from the melanogaster and obscura groups. The genus Zaprionus and the subgenera Dorsilopha and Hirtodrosophila appear as branches of a prolific “bush” that also embraces the numerous species of the subgenus Drosophila. The Sod results corroborate in many, but not all, respects Throckmorton's (King, R.C. (ed) Handbook of Genetics. Plenum Press, New York, pp. 421–469, 1975) phylogeny; are inconsistent in some important ways with Grimaldi's (Bull. Am. Museum Nat. Hist. 197:1–139, 1990) cladistic analysis; and also are inconsistent with some inferences based on mitochondrial DNA data. The Sod results manifest how, in addition to the information derived from nucleotide sequences, structural features (i.e., the deletion of an intron) can help resolve phylogenetic issues. Correspondence requests to: F. J. Ayala     

ON THE ORIGIN OF INCIPIENT REPRODUCTIVE ISOLATION: THE CASE OF DROSOPHILA ALBOMICANS AND D. NASUTA

The nasuta subgroup of Drosophila consists of 12 known species classified within the immigrans group. D. nasuta and D. albomicans are two sibling species widely distributed throughout the Indo-Pacific tropics, which, although morphologically indistinguishable, have different meta-phase-chromosome configurations: chromosomes X and 3 are attached in D. albomicans, so that about 60% of its genes are sex-linked. Our experiments show that, at least in the laboratory, there is no sexual, mechanical, or gametic isolation between the two species. There is, however, hybrid “breakdown” expressed in three ways: 1) reduction in the number of F₂ hybrids produced per culture; 2) reduction in the fertility of F₂ (males) and F₃ (males and females) hybrid progenies; and 3) abnormal sex ratios in the progenies of crosses between strains of certain localities. In experimental populations, the karyotypes of both species are still present in substantial frequencies after 20 generations, although the frequencies of the two karyotypes vary depending on the geographic origin of the strains. Our results support the hypothesis that, in allopatry, the evolution of postzygotic isolation precedes that of prezygotic isolation. The mtDNA is polymorphic in both D. nasuta and D. albomicans and fairly similar between them. Assuming typical rates of mtDNA evolution, the two species would have diverged from each other about 500,000 years ago, whereas the African and Indian populations of D. nasuta (considered to be different subspecies by some authors) might have diverged some 350,000 years ago.