Nucleotide sequence of a chromosomal mercury resistance determinant from a Bacillus sp. with broad-spectrum mercury resistance.

A 13.5-kilobase HindIII fragment, bearing an intact mercury resistance (mer) operon, was isolated from chromosomal DNA of broad-spectrum mercury-resistant Bacillus sp. strain RC607 by using as a probe a clone containing the mercury reductase (merA) gene. The new clone, pYW33, expressed broad-spectrum mercury resistance both in Escherichia coli and in Bacillus subtilis, but only in B. subtilis was the mercuric reductase activity inducible. Sequencing of a 1.8-kilobase mercury hypersensitivity-producing fragment revealed four open reading frames (ORFs). ORF1 may code for a regulatory protein (MerR). ORF2 and ORF4 were associated with cellular transport function and the hypersensitivity phenotype. DNA fragments encompassing the merA and the merB genes were sequenced. The predicted Bacillus sp. strain RC607 MerA (mercuric reductase) and MerB (organomercurial lyase) were similar to those predicted from Staphylococcus aureus plasmid pI258 (67 and 73% amino acid identities, respectively); however, only 40% of the amino acid residues of RC607 MerA were identical to those of the mercuric reductase from gram-negative bacteria. A 69-kilodalton polypeptide was isolated and identified as the merA gene product by examination of its amino-terminal sequence.     

Formation of yeast mitochondria III: Biochemical properties of mitochondria isolated from a cytoplasmic petite mutant

A number of biochemical properties of mitochondria from a cytoplasmic petite mutant ofSaccharomyces cerevisiae with an extremely high adenine plus thymine content have been studied.When such particles are isolated by means of standard procedures developed for use with wild-type yeasts they are grossly contaminated by non-mitochondrial membrane fragments. Further enrichment of mitochondria is achieved by non-equilibrium centrifugation in sucrose gradients.Throughout this purification procedure the particles can be shown to retain an outer limiting, as well as a non-cristate inner membrane. In many of their morphological and physical features (size, shape, buoyant density) they resemble mitochondria isolated from the wild type.Although enzymes of the respiratory chain are absent from the mutant particles, their content ofl-malate dehydrogenase, NADP-dependent isocitrate dehydrogenase, and ATPase is comparable to that found in the wild type. The mitochondrial ATPase in this mutant strain is cold stable, oligomycin insensitive, Dio-9 sensitive, and susceptible to inhibition by the F₁ inhibitor of beef heart. The enzyme can be rendered cold labile by its detachment from the membrane, followed by fractionation with protamine sulfate and ammonium sulfate.The existence of mutant particles that are incapable of function in oxidation and phosphorylation but resemble their functional homologues in many other ways raises the possibility that mitochondria are required in the cellular economy for purposes not directly linked to oxidative phosphorylation and electron transport. This hypothesis has led us to suggest that, contrary to models currently under discussion, mitochondria did not evolve as a consequence of endosymbiosis. We propose as an alternative that the mitochondrial organelle evolved as a means of improvement of existing subcellular structures in the primordial (perhaps eukaryotic) cell. Partial autonomy may thus constitute a relatively recent modification; the present-day mitochondrial genome had its origin in nuclear DNA and may have been amplified in a manner not unlike the amplification of ribosomal RNA cistrons in developing oocytes ofXenopus.Supported by Research Grant GM 12228 from the National Institute of General Medical Science, National Institutes of Health, U.S. Public Health Service.Recipient of a Public Health Service Career Award No. GM 05060 from the Institute of General Medical Sciences.     

Structural requirements for mitochondrial mutagenesis

Ethidium bromide (3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide) and euflavine (3, 6-diamino-10-methylacridinium chloride) are superficially similar in structure and ability to intercalate into DNA. However, they exhibit qualitative differences in their ability to bring about a mitochondrial mutation (ρ⁺ → ρ^?) in Saccharomyces cerevisiae. This investigation tried to establish and compare the essential structural prerequisites in three series of planar, heterocyclic dyes: the phenanthridines (P series), the acridines (A series), and molecules with different heteroatoms related to acridines (X series). Compounds capable of bringing about the mutation in the complete absence of growth and energy sources are restricted to di-primary amines in the P series: quaternization of the ring nitrogen, and an aromatic side chain at C-6 also appear essential. Compounds in the A series are mutagenic only with growing cells; quaternization (C₁ through C₄) is essential. The 10-allyl derivative is unusual; it is highly effective even in buffer supplemented only with an energy source. The results are interpreted in terms of a model that requires interaction of the mutagen with the mitochondrial inner membrane as well as with its DNA.     

Studies on cytochrome oxidase. Partial resolution of enzymes containing seven or six subunits, from yeast and beef heart, respectively.

Highly active, essentially homogeneous, preparations of ferrocytochrome c oxidase (EC 1.9.3.1) have been obtained from both yeast and beef heart by extraction with cholate, fractionation with ammonium sulfate, and replacement of cholate by Tween 20. The molecular weights of the resultant proteins equal 260 +/- 23 X 10(3) and 205 +/- 10(3); they contain seven and six different polypeptide subunits, respectively, all in equimolar amounts, with apparent molecular weights of 42.4, 34.1, 24.7, 14.6, 14.6, 12.3, 10.6 X 10(3), and 47.5, 20.4, 14.5, 14.5, 13.0, 11.0 X 10(3), respectively. By means of apolar chromatography on L-leucine coupled to agarose these enzymes can be stripped of their largest subunit(s) resulting in preparations with molecular weights of 170 +/- 17 X 10(3) and 124 +/- 20 X 10(3), and containing only five polypeptides, with the largest remaining one (molecular weight congruent to 20 X 10(3)) present in less than stoichiometric amounts. This interconversion and subunit removal has been monitored by exclusion chromatography, four systems of acrylamide gel electrophoresis--some with the protein labeled with 125I under denaturing conditions--isoelectric focusing, and hydrodynamic methods. It has virtually no effect on heme a and copper content and on the catalytic parameters of the enzymes. We conclude that subunits I and II in enzymes from fungal, and subunit I in those from animal, sources are dispensable for the catalysis of the oxidation of ferrocytochrome c by, and are probably not essential for the attatchment of prosthetic groups to, these proteins.     

Roles for modularity and constraint in the evolution of cranial diversity among Anolis lizards

Complex organismal structures are organized into modules, suites of traits that develop, function, and vary in a coordinated fashion. By limiting or directing covariation among component traits, modules are expected to represent evolutionary building blocks and to play an important role in morphological diversification. But how stable are patterns of modularity over macroevolutionary timescales? Comparative analyses are needed to address the macroevolutionary effect of modularity, but to date few have been conducted. We describe patterns of skull diversity and modularity in Caribbean Anolis lizards. We first diagnose the primary axes of variation in skull shape and then examine whether diversification of skull shape is concentrated to changes within modules or whether changes arose across the structure as a whole. We find no support for the hypothesis that cranial modules are conserved as species diversify in overall skull shape. Instead we find that anole skull shape and modularity patterns independently converge. In anoles, skull modularity is evolutionarily labile and may reflect the functional demands of unique skull shapes. Our results suggest that constraints have played little role in limiting or directing the diversification of head shape in Anolis lizards.     

Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability

Cocultures of two human cell lines, Caco-2 and HT29-MTX mucus-producing cells, have been incorporated into an in vitro digestion/cell culture model used to predict iron bioavailability. A range of different foods were subjected to in vitro digestion, and iron bioavailability from digests was assessed with Caco-2, Caco-2 overlaid with porcine mucin, HT29-MTX or cocultures of Caco-2 and HT29-MTX at varying ratios. It was found that increasing the ratio of HT29-MTX cells decreased the amount of ferritin formed and resulted in an overall decline in the ability of the model to detect differences in iron bioavailability. At the physiologically relevant ratios of 90% Caco-2/10% HT29-MTX and 75% Caco-2/25% HT29-MTX, however, a mucus layer completely covered the cell monolayer and the in vitro digestion model was nearly as responsive to changes in sample iron bioavailability as pure Caco-2 cultures. The in vitro digestion/Caco-2 cell culture model correlates well with human iron bioavailability studies, but, as mucus appears to play a role in iron absorption, the addition of a physiologically realistic mucus layer and goblet-type cells to this model may give more accurate iron bioavailability predictions.     

Characterization of a gastrointestinal tract microscale cell culture analog used to predict drug toxicity

The lining of the gastrointestinal (GI) tract is the largest surface exposed to the external environment in the human body. One of the main functions of the small intestine is absorption, and intestinal absorption is a route used by essential nutrients, chemicals, and pharmaceuticals to enter the systemic circulation. Understanding the effects of digestion on a drug or chemical, how compounds interact with and are absorbed through the small intestinal epithelium, and how these compounds affect the rest of the body is critical for toxicological evaluation. Our goal is to create physiologically realistic in vitro models of the human GI tract that provide rapid, inexpensive, and accurate predictions of the body's response to orally delivered drugs and chemicals. Our group has developed an in vitro microscale cell culture analog (µCCA) of the GI tract that includes digestion, a mucus layer, and physiologically realistic cell populations. The GI tract µCCA, coupled with a multi‐chamber silicon µCCA representing the systemic circulation, is described and challenged with acetaminophen. Proof of concept experiments showed that acetaminophen passes through and is metabolized by the in vitro intestinal epithelium and is further metabolized by liver cells, resulting in liver cell toxicity in a dose‐dependent manner. The µCCA response is also consistent with in vivo measurements in mice. The system should be broadly useful for studies on orally delivered drugs or ingestion of chemicals with potential toxicity. Biotechnol. Bioeng. 2009; 104: 193–205 © 2009 Wiley Periodicals, Inc.