An oxygen-dependent coproporphyrinogen oxidase encoded by the hemF gene of Salmonella typhimurium.

The 8th step in the 10-step heme biosynthetic pathway of Salmonella typhimurium is the oxidation of coproporphyrinogen III to protoporphyrinogen IX. On the basis of genetic studies, we have suggested that this reaction may be catalyzed by either of two different enzymes, an oxygen-dependent one encoded by hemF or an oxygen-independent enzyme encoded by hemN. Here, we report the cloning of the S. typhimurium hemF gene and its DNA sequence. The predicted amino acid sequence of the HemF protein is 44% identical to that of the coproporphyrinogen oxidase encoded by the yeast HEM13 gene. The wild-type S. typhimurium strain LT-2 produces an oxygen-dependent coproporphyrinogen oxidase activity detectable in crude extracts, which is not found in hemF mutants and is overproduced in strains carrying the hemF gene on a multicopy plasmid. the hemF gene is the second gene in an operon with an upstream gene with an unknown function, whose amino acid sequence suggests a relation to amidases involved in cell wall synthesis or remodeling. The upstream gene and hemF are cotranscribed from a promoter which was mapped by primer extension. A weaker, hemF-specific promoter is inferred from the behavior of an omega-Cm insertion mutation in the upstream gene. Although this insertion decreases expression of beta-galactosidase about 7.5-fold when placed upstream of a hemF-lacZ operon fusion, it still allows sufficient HemF expression from an otherwise wild-type construct to confer a Hem+ phenotype. The hemF operon is transcribed clockwise with respect to the genetic map.     

The genes required for heme synthesis in Salmonella typhimurium include those encoding alternative functions for aerobic and anaerobic coproporphyrinogen oxidation.

Insertion mutagenesis has been used to isolate Salmonella typhimurium strains that are blocked in the conversion of 5-aminolevulinic acid (ALA) to heme. These mutants define the steps of the heme biosynthetic pathway after ALA. Insertions were recovered at five unlinked loci: hemB, hemCD, and hemE, which have been mapped previously in S. typhimurium, and hemG and hemH, which have been described only for Escherichia coli. No other simple hem mutants were found. However, double mutants are described that are auxotrophic for heme during aerobic growth and fail to convert coproporphyrinogen III to protoporphyrinogen IX. These mutant strains are defective in two genes, hemN and hemF. Single mutants defective only in hemN require heme for anaerobic growth on glycerol plus nitrate but not for aerobic growth on glycerol. Mutants defective only in hemF have no apparent growth defect. We suggest that these two genes encode alternative forms of coproporphyrinogen oxidase. Anaerobic heme synthesis requires hemN function, while either hemN or hemF is sufficient for aerobic heme synthesis. These phenotypes are consistent with the requirement of a well-characterized class of coproporphyrinogen oxidase for molecular oxygen.     

Porphyrin and corrinoid mutants of Bacillus subtilis.

Porphyrin auxotrophs of Bacillus subtilis can be divided into two groups. Strains belonging to the first group (hemA, hemB, or hemC) are not able to synthesize or metabolize porphobilinogen. These strains require cysteine, cystine, and methionine, respectively. Traces of aminolevulinic acid, in a hemin-containing medium, can replace the cysteine requirement in a mutant lacking aminolevulinic acid synthetase. In bacteria belonging to the second group (hemE, hemF, or hemG), porphyrin biosynthesis is blocked at later steps, and the amino acids mentioned above are not required. It is of interest that both the activity of ribonucleotide reductase and the amount of vitamin B12 were significantly lower in the first group. The addition of vitamin B12 to the medium did not promote the growth of strains examined. We assume that porphobilinogen deaminase is essential for the synthesis of corrinoids.     

Oxygen-dependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese

During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.     

Zcchc8 is a glycogen synthase kinase-3 substrate that interacts with RNA-binding proteins

Phosphorylation of c-Myc on threonine 58 (T58) stimulates its degradation by the Fbw7-SCF ubiquitin ligase. We used a phosphorylation-specific antibody raised against the c-Myc T58 region to attempt to identify other proteins regulated by the Fbw7 pathway. We identified two predominant proteins recognized by this antibody. The first is Ebna1 binding protein 2, a nucleolar protein that, in contrast with a previous report, is likely responsible for the nucleolar staining exhibited by this antibody. The second is Zcchc8, a nuclear protein that is highly phosphorylated in cells treated with nocodazole. We show that Zcchc8 is directly phosphorylated by GSK-3 in vitro and that GSK-3 inhibition prevents Zcchc8 phosphorylation in vivo. Moreover, we found that Zcchc8 interacts with proteins involved in RNA processing/degradation. We suggest that Zcchc8 is a GSK-3 substrate with a role in RNA metabolism.     

The origin of plastids

It is generally accepted that plastids first arose by acquisition of photosynthetic prokaryotic endosymbionts by non-photosynthetic eukaryotic hosts. It is also accepted that photosynthetic eukaryotes were acquired on several occasions as endosymbionts by non-photosynthetic eukaryote hosts to form secondary plastids. In some lineages, secondary plastids were lost and new symbionts were acquired, to form tertiary plastids. Most recent work has been interpreted to indicate that primary plastids arose only once, referred to as a 'monophyletic' origin. We critically assess the evidence for this. We argue that the combination of Ockham's razor and poor taxon sampling will bias studies in favour of monophyly. We discuss possible concerns in phylogenetic reconstruction from sequence data. We argue that improved understanding of lineage-specific substitution processes is needed to assess the reliability of sequence-based trees. Improved understanding of the timing of the radiation of present-day cyanobacteria is also needed. We suggest that acquisition of plastids is better described as the result of a process rather than something occurring at a discrete time, and describe the 'shopping bag' model of plastid origin. We argue that dinoflagellates and other lineages provide evidence in support of this.     

Predicting the unpredictable: transmission of drug-resistant HIV

We use a mathematical model to understand (from 1996 to 2001) and to predict (from 2001 to 2005) the evolution of the epidemic of drug-resistant HIV in San Francisco. We predict the evolutionary trajectories for 1,000 different drug-resistant strains with each strain having a different fitness relative to a drug-sensitive strain. We calculate that the current prevalence of resistance is high, and predict it will continue to rise. In contrast, we calculate that transmission of resistance is currently low, and predict it will remain low. We show that the epidemic of resistance is being generated mainly by the conversion of drug-sensitive cases to drug-resistant cases, and not by the transmission of resistant strains. We also show that transmission of resistant strains has not increased the overall number of new HIV infections. Our results indicate that transmission of resistant strains is, and will remain, a relatively minor public health problem.     

Cholesterol-Dependent Bending Energy Is Important in Cholesterol Distribution of the Plasma Membrane

We consider the plasma membrane that contains a cholesterol molar fraction of 0.4 and ask how that cholesterol is distributed between the two leaves. Because of the rapid flip-flop of cholesterol between leaves, we assume that its distribution is determined by the equality of its chemical potentials in the two leaves. When we consider only the contributions of entropy and interactions to the cholesterol chemical potential in our model system, we find, not surprisingly, that the cholesterol is mostly in the outer leaf because of the strong attraction between cholesterol and sphingomyelin (SM), which is predominantly in that leaf. We find 72% there. We then include the contribution from the bending energy in each leaf that must be overcome to join the leaves in a flat bilayer. The product of bending modulus and spontaneous curvature is obtained from simulation. We find that the addition of cholesterol to the outer leaf reduces the spontaneous curvature, which is initially positive, until it passes through zero when the molar fraction of cholesterol in the outer leaf is 0.28. Additional cholesterol is driven toward the inner leaf by the sphingomyelin phosphatidylcholine mixture. This is resisted by the bending energy contribution to the inner leaf. We find, again by simulation, that the addition of cholesterol monotonically increases the magnitude of the spontaneous curvature of the inner leaf, which is negative. This increases its bending energy. We conclude that, as a result of these competing effects, the percentage of cholesterol in the outer leaf is reduced to ∼63 ± 6%.     

Global stability of population models

Local stability seems to imply global stability for population models. To investigate this claim, we formally define apopulation model. This definition seems to include the one-dimensional discrete models now in use. We derive a necessary and sufficient condition for the global stability of our defined class of models. We derive an easily testable sufficient condition for local stability to imply global stability. We also show that if a discrete model is majorized by one of these stable population models, then the discrete model is globally stable. We demonstrate the utility of these theorems by using them to prove that the regions of local and global stability coincide for six models from the literature. We close by arguing that these theorems give a method for demonstrating global stability that is simpler and easier to apply than the usual method of Liapunov functions.     

A model for anteroposterior patterning of the vertebrate limb based on sequential long- and short-range Shh signalling and Bmp signalling

It has been proposed that digit identity in chick limb bud is specified in a dose-dependent fashion by a long-range morphogen, produced by the polarising region. One candidate is Sonic hedgehog (Shh) protein, but it is not clear whether Shh acts long or short range or via Bmps. Here we dissect the relationship between Shh and Bmp signalling. We show that Shh is necessary not only for initiating bmp2 expression but also for sustaining its expression during the period when additional digits are being specified. We also show that we can reproduce much of the effect of Shh during this period by applying only Bmp2. We further demonstrate that it is Bmps that are responsible for digit specification by transiently adding Noggin or Bmp antibodies to limbs treated with Shh. In such limbs, multiple additional digits still form but they all have the same identity. We also explored time dependency and range of Shh signalling by examining ptc expression. We show that high-level ptc expression is induced rapidly when either Shh beads or polarising regions are grafted to a host limb. Furthermore, we find that high-level ptc expression is first widespread but later more restricted. All these data lead us to propose a new model for digit patterning. We suggest that Shh initially acts long range to prime the region of the limb competent to form digits and thus control digit number. Then later, Shh acts short range to induce expression of Bmps, whose morphogenetic action specifies digit identity.     

The force exerted by a muscle cross-bridge depends directly on the strength of the actomyosin bond

Myosin produces force in a cyclic interaction, which involves alternate tight binding to actin and to ATP. We have investigated the energetics associated with force production by measuring the force generated by skinned muscle fibers as the strength of the actomyosin bond is changed. We varied the strength of the actomyosin bond by addition of a polymer that promotes protein-protein association or by changing temperature or ionic strength. We estimated the free energy available to generate force by measuring isometric tension, as the free energy of the states that precede the working stroke are lowered with increasing phosphate. We found that the free energy available to generate force and the force per attached cross-bridge at low [Pi] were both proportional to the free energy available from the formation of the actomyosin bond. We conclude that the formation of the actomyosin bond is involved in providing the free energy driving the production of isometric tension and mechanical work. Because the binding of myosin to actin is an endothermic, entropically driven reaction, work must be performed by a "thermal ratchet" in which a thermal fluctuation in Brownian motion is captured by formation of the actomyosin bond.     

Studies on the interaction of the human c-myc protein with cell nuclei: p62c-myc as a member of a discrete subset of nuclear proteins

We have analyzed the localization of the human c-myc product (p62c-myc) at steady state in cells by immunoprecipitation and immunoblotting. We show that p62c-myc is extracted from nuclei by mild salt concentrations (below 200 mM), without affecting gross nuclear structure or causing extraction of major chromatin components. We observe no association between p62c-myc and the nuclear matrix. We also demonstrate that p62c-myc is a member of a discrete subset of nuclear proteins that are all rendered irreversibly insoluble in situ by exposure of isolated nuclei to physiological temperatures (37 degrees C). p62c-myc is sequestered into a similar insoluble complex in cells that have been subjected to heat shock. Finally, we show that avian v-myc and v-myb proteins in isolated nuclei also become insoluble after exposure to temperatures above 37 degrees C. We discuss the possible implications of these results.     

An alternative route to coloniality in the bearded tit: females pursue extra-pair fertilizations

We combine behavioral studies with DNA-fingerprinting of a populationof bearded tits Panurus biarmicus and test the idea that thepursuit of extra-pair copulations by females acts as selectivepressure that may contribute to the formation of colonies inthis socially monogamous species. We show that the rate of extra-pairpaternity in colonial breeding pairs is very high, whereas noextra-pair chicks were detected in solitary nests. We foundno cases of extra-pair maternity and intraspecific brood parasitismin solitary nests, whereas it did occur in dense breeding situations.We further found no difference in the quality of males betweencolonial and solitary individuals. However, colonial femaleswere of significantly higher quality in terms of body size andcondition. We propose that high-quality females settle in coloniesto increase their opportunities to adjust the choice of theirsocial partner by obtaining extra-pair copulations. We suggestthat colony formation enables high-quality females to incitemale-male competition for extra-pair copulations and consequentlysecure extra-pair fertilizations by high-quality males and thathigh-density nesting is controlled by female bearded tits