The sedimentation behaviour of ribonuclease-active and -inactive ribosomes from bacteria

1. The `30s' and `50s' ribosomes from ribonuclease-active (Escherichia coli B) and -inactive (Pseudomonas fluorescens and Escherichia coli MRE600) bacteria have been studied in the ultracentrifuge. Charge anomalies were largely overcome by using sodium chloride–magnesium chloride solution, I 0·16, made 0–50mm with respect to Mg²⁺. 2. Differentiation of enzymic and physical breakdown at Mg²⁺ concentrations less than 5mm was made by comparing the properties of E. coli B and P. fluorescens ribosomes. 3. Ribonuclease-active ribosomes alone showed a transformation of `50s' into 40–43s components. This was combined with the release of a small amount of `5s' material which may be covalently bound soluble RNA. Other transformations of the `50s' into 34–37s components were observed in both ribonuclease-active and -inactive ribosomes at 1·0–2·5mm-Mg²⁺, and also with E. coli MRE600 when EDTA (0·2mm) was added to a solution in 0·16m-sodium chloride. 4. Degradation of ribonuclease-active E. coli B ribosomes at Mg²⁺ concentration 0·25mm or less was coincident with the formation of 16s and 21s ribonucleoprotein in P. fluorescens, and this suggested that complete dissociation of RNA from protein was not an essential prelude to breakdown of the RNA by the enzyme. 5. As high Cs⁺/Mg²⁺ ratios cause ribosomal degradation great care is necessary in the interpretation of equilibrium-density-gradient experiments in which high concentrations of caesium chloride or similar salts are used. 6. The importance of the RNA moiety in understanding the response of ribosomes to their ionic environment is discussed.     

Erythrocruorin of Marphysa sanguinea. Isolation of some physical, physicochemical and other properties

1. The polychaete worm Marphysa sanguinea has a circulating erythrocruorin of mol.wt. about 2·4×10⁶ (S⁰_20,w 58·2s, D_20,w 2·06×10⁻⁷ cm.²/sec). This is the predominant form existing at pH 6–8 and (non-protein) I 0·10–0·21, and also at approx. pH 6·7 and I 0·15–3·00. 2. The pigment contains 2·24% of protohaem. 3. The 58s protein has an electrophoretic mobility of 8·08×10⁻⁵ cm.²/v/sec. at pH 8·12, I 0·21 and 0°. The isoelectric point of suspended particles is 4·63 at I 0·16 and 21·5°. 4. At very low ionic strength and pH 6·7 (unbuffered) the 58s pigment associates reversibly to 97s and 150s forms, which are probably dimer and tetramer species. 5. At pH 10·0 and I 0·025, it dissociates irreversibly to give a small amount of 2–4s non-haem-containing protein and much 9s haem-enriched protein. These and the 58s pigment may correspond to structures found in Levin's (1963) electron-microscope studies of other erythrocruorins. 6. Absorption spectra of the 58s oxygenated erythrocruorin and the deoxygenated and carbon monoxide derivatives have been obtained.     

Computing folding pathways between RNA secondary structures

Given an RNA sequence and two designated secondary structures A, B, we describe a new algorithm that computes a nearly optimal folding pathway from A to B. The algorithm, RNAtabupath, employs a tabu semi-greedy heuristic, known to be an effective search strategy in combinatorial optimization. Folding pathways, sometimes called routes or trajectories, are computed by RNAtabupath in a fraction of the time required by the barriers program of Vienna RNA Package. We benchmark RNAtabupath with other algorithms to compute low energy folding pathways between experimentally known structures of several conformational switches. The RNApathfinder web server, source code for algorithms to compute and analyze pathways and supplementary data are available at http://bioinformatics.bc.edu/clotelab/RNApathfinder.     

Estimating Selection Intensity on Synonymous Codon Usage in a Nonequilibrium Population

Codon usage bias is the nonrandom use of synonymous codons for the same amino acid. Most population genetic models of codon usage evolution assume that the population is at mutation–selection–drift equilibrium. Natural populations, however, frequently deviate from equilibrium, often because of recent demographic changes. Here, we construct a matrix model that includes the effects of a recent change in population size on estimates of selection on preferred vs. unpreferred codons. Our results suggest that patterns of synonymous polymorphisms affecting codon usage can be quite erratic after such a change; statistical methods that fail to take demographic effects into account can then give incorrect estimates of important parameters. We propose a new method that can accurately estimate both demographic and codon usage parameters. The method also provides a simple way of testing for the effects of covariates such as gene length and level of gene expression on the intensity of selection, which we apply to a large Drosophila melanogaster polymorphism data set. Our analyses of twofold degenerate codons reveal that (i) selection acts in favor of preferred codons, (ii) there is mutational bias in favor of unpreferred codons, (iii) shorter genes and genes with higher expression levels are under stronger selection, and (iv) there is little evidence for a recent change in population size in the Zimbabwe population of D. melanogaster.CODONS specifying the same amino acid are called synonymous codons. These are often used nonrandomly, with some codons appearing more frequently than others. This biased usage of synonymous codons has been found in many organisms such as Drosophila, yeast, and bacteria (Ikemura 1985; Duret and Mouchiroud 1999; Hershberg and Petrov 2008). Conventionally, synonymous codons for a given amino acid are divided into two classes: preferred and unpreferred codons (Ikemura 1985; Akashi 1994; Duret and Mouchiroud 1999). Several observations indicate that codon usage is affected by natural selection. First, in species with codon usage bias, preferred codons generally correspond to the most abundant tRNA species (Ikemura 1981). Second, highly expressed genes usually have higher codon usage bias than genes with low expression (Sharp and Li 1986; Duret and Mouchiroud 1999; Hey and Kliman 2002). Third, the synonymous substitution rate of a gene has been shown to be negatively correlated with its degree of codon usage bias (Sharp and Li 1986; Bierne and Eyre-Walker 2006). The most commonly cited explanations of the apparent fitness differences between preferred and unpreferred codons are selection for translation efficiency, translational accuracy, and mRNA stability (Ikemura 1985; Eyre-Walker and Bulmer 1993; Akashi 1994; Drummond et al. 2005). Recently, it has been proposed that exon splicing also affects codon usage bias (Warnecke and Hurst 2007).From a population genetics perspective, the extent of codon usage bias is ultimately a product of the joint effects of mutation, selection, genetic drift, recombination, and demographic history. The Li–Bulmer model of drift, selection, and reversible mutation between preferred and unpreferred codons at a site is the most widely used model (Li 1987; Bulmer 1991; McVean and Charlesworth 1999). Applications of this model generally assume that the population is at mutation–selection–drift equilibrium. However, empirical studies have suggested that changes in the strengths of various driving forces may not be unusual. For example, in Drosophila melanogaster, there is evidence that the population size (Li and Stephan 2006; Thornton and Andolfatto 2006; Keightley and Eyre-Walker 2007; Stephan and Li 2007), recombinational landscape (Takano-Shimizu 1999), and mutational process (Takano-Shimizu 2001; Kern and Begun 2005) may have changed significantly over the species'' evolutionary history.Such changes cause departures from equilibrium. Theoretical models show that it takes a very long time, proportional to the reciprocal of the mutation rate, for the population to approach a new equilibrium state (Tachida 2000; Comeron and Kreitman 2002). Before reaching equilibrium, the population often shows counterintuitive patterns of evolution (Eyre-Walker 1997; Takano-Shimizu 1999, 2001; Comeron and Kreitman 2002; Comeron and Guthrie 2005; Charlesworth and Eyre-Walker 2007). Despite these theoretical results, details of the patterns of polymorphism and substitution rates following a recent change in population size, and their effects on estimates of strength of selection, have not been determined.The above findings point to the importance of incorporating nonequilibrium factors into the study of codon usage bias. To this end, we extend the Li–Bulmer model to allow population size to vary over time, by representing the evolutionary process by a transition matrix. By analyzing this matrix model, we show that a recent change in population size can result in erratic patterns of codon usage and that methods failing to take into account these demographic effects can give false estimates of the intensity of selection.To solve these problems, we propose a new method, which does not require polarizing ancestral vs. derived states using outgroup data (cf. Cutter and Charlesworth 2006), but requires only knowledge of preferred vs. unpreferred states defined by patterns of codon usage. We use information on both polymorphic and fixed sites, which enables both mutational bias and the strength of selection to be estimated, in contrast to previous methods that use information on polymorphisms alone. Simulations indicate that this method can accurately estimate both demographic and codon usage parameters and can distinguish between selection and demography. We use the new method to analyze a large D. melanogaster polymorphism data set (Shapiro et al. 2007) and find evidence for natural selection on synonymous codons. We use our approach to show that genes with shorter coding sequences and higher levels of expression are under significantly stronger selection than longer genes with lower expression.     

CO(2) Metabolism in Corn Roots. III. Inhibition of P-enolpyruvate Carboxylase by l-malate

Phosphoenolpyruvate carboxylase was purified from corn root tips about 80-fold by centrifugation, ammonium sulfate fractionation, and anion exchange and gel filtration chromatography. The resulting preparation was essentially free from malate dehydrogenase, isocitrate dehydrogenase, malate enzyme, NADH oxidase, and pyruvate kinase activity. Kinetic analysis indicated that l-malate was a noncompetitive inhibitor of P-enolpyruvate carboxylase with respect to P-enolpyruvate (K_I = 0.8 mm). d-Malate, aspartate, and glutamate inhibited to a lesser extent; succinate, fumarate, and pyruvate did not inhibit. Oxaloacetate was also a noncompetitive inhibitor of P-enolpyruvate carboxylase with an apparent K_I of 0.4 mm. A comparison of oxaloacetate and l-malate inhibition suggested that the mechanisms of inhibition were different. These data indicated that l-malate may regulate CO₂ fixation in corn root tips by a feedback or end product type of inhibition.     

Gorilla Mothers Also Matter! New Insights on Social Transmission in Gorillas (Gorilla gorilla gorilla) in Captivity

The present paper describes two distinct behaviors relating to food processing and communication that were observed in a community of five separately housed groups of lowland gorillas (Gorilla gorilla gorilla) in captivity during two study periods one decade apart: (1) a food processing technique to separate wheat from chaff, the so-called puff-blowing technique; and (2) a male display used to attract the attention of visitors, the so-called throw-kiss-display. We investigated (a) whether the behaviors were transmitted within the respective groups; and if yes, (b) their possible mode of transmission. Our results showed that only the food processing technique spread from three to twenty-one individuals during the ten-year period, whereas the communicative display died out completely. The main transmission mode of the puff-blowing technique was the mother-offspring dyad: offspring of puff-blowing mothers showed the behavior, while the offspring of non- puff-blowing mothers did not. These results strongly support the role mothers play in the acquisition of novel skills and vertical social transmission. Furthermore, they suggest that behaviors, which provide a direct benefit to individuals, have a high chance of social transmission while the loss of benefits can result in the extinction of behaviors.     

The Drosophila Planar Polarity Proteins Inturned and Multiple Wing Hairs Interact Physically and Function Together

The conserved frizzled (fz) pathway regulates planar cell polarity in both vertebrate and invertebrate animals. This pathway has been most intensively studied in the wing of Drosophila, where the proteins encoded by pathway genes all accumulate asymmetrically. Upstream members of the pathway accumulate on the proximal, distal, or both cell edges in the vicinity of the adherens junction. More downstream components including Inturned and Multiple Wing Hairs accumulate on the proximal side of wing cells prior to hair initiation. The Mwh protein differs from other members of the pathway in also accumulating in growing hairs. Here we show that the two Mwh accumulation patterns are under different genetic control with the early proximal accumulation being regulated by the fz pathway and the latter hair accumulation being largely independent of the pathway. We also establish recruitment by proximally localized Inturned to be a putative mechanism for the localization of Mwh to the proximal side of wing cells. Genetically inturned (in) acts upstream of mwh (mwh) and is required for the proximal localization of Mwh. We show that Mwh can bind to and co-immunoprecipitate with Inturned. We also show that these two proteins can function in close juxtaposition in vivo. An In∷Mwh fusion protein provided complete rescue activity for both in and mwh mutations. The fusion protein localized to the proximal side of wing cells prior to hair formation and in growing hairs as expected if protein localization is a key for the function of these proteins.THE frizzled (fz) signaling pathway regulates tissue planar cell polarity (PCP) in the epidermis of both vertebrate and invertebrate animals (Lawrence et al. 2007; Montcouquiol 2007; Wang and Nathans 2007; Zallen 2007). PCP is dramatic in the cuticle of insects such as Drosophila, which is decorated with arrays of hairs and sensory bristles.The genetic basis for tissue polarity has been most extensively studied in the fly wing (Wong and Adler 1993). The Planar Polarity (PCP) genes of the fz pathway (also known as the core PCP genes), the planar polarity effector (PPE) genes and the multiple wing hairs (mwh) gene encode key components that regulate planar polarity in the wing. fz, disheveled (dsh), prickle/spiny leg (pk/sple), Van Gogh (Vang) (aka strabismus), starry night (stan) (aka flamingo) and diego (dgo) are members of the PCP group (Vinson and Adler 1987; Wong and Adler 1993; Taylor et al. 1998; Wolff and Rubin 1998; Chae et al. 1999; Gubb et al. 1999; Usui et al. 1999). A distinctive feature of these genes is that their protein products accumulate asymmetrically on the distal (Fz, Dsh, and Dgo) (Axelrod 2001; Feiguin et al. 2001; Shimada et al. 2001; Strutt 2001), proximal (Vang, Pk)(Tree et al. 2002; Bastock et al. 2003), or both distal and proximal (Stan) (Usui et al. 1999) sides of wing cells. These genes/proteins act as a functional group and are corequirements for the asymmetric accumulation of the others.The PPE includes inturned (in), fuzzy (fy), and fritz (frtz) (Park et al. 1996; Collier and Gubb 1997; Collier et al. 2005). These genes are thought to function downstream of the PCP genes and the proteins encoded by these genes also accumulate asymmetrically in wing cells (Adler et al. 2004; Strutt and Warrington 2008). As is the case for the PCP genes, the PPE genes/proteins also appear to be a functional group and to be corequirements for the asymmetric accumulation of the others. Several observations support the hypothesis that the PPE genes are essential downstream effectors of the PCP genes. The earliest appreciation of this came from careful observations of the mutant phenotypes. A common feature of mutations in all of these genes is that they do not result in a randomization of hair polarity, but rather in a similar complicated and abnormal stereotypic pattern (Gubb and Garcia-Bellido 1982; Adler et al. 2000). That the abnormal patterns were so similar suggested that these genes all functioned in the same process (Wong and Adler 1993). The mutant phenotypes differed in that the vast majority of PCP mutant wing cells form a single hair, while many PPE mutant wing cells form two or three hairs. Mutations in PPE genes are epistatic to both loss- and gain-of-function mutations in PCP genes (Wong and Adler 1993; Lee and Adler 2002). Further evidence that the PPE genes function downstream of the PCP genes comes from the analysis of protein localization. PPE gene function is not needed for the proper asymmetric localization of PCP proteins (Usui et al. 1999; Strutt 2001; Tree et al. 2002; Collier et al. 2005) but in contrast PCP gene function is essential for the asymmetric accumulation of PPE proteins (Adler et al. 2004; Strutt and Warrington 2008). Further, the PCP genes/proteins instruct the localization of the PPE proteins (Adler et al. 2004).The multiple-wing-hairs (mwh) gene is thought to function downstream of both the PCP and PPE genes (Wong and Adler 1993). This conclusion comes from analyses that are similar to those that established that the PPE genes function downstream of the PCP genes. The overall hair polarity pattern of mwh mutant wings shares the same complicated and abnormal stereotypic hair polarity pattern seen in PCP and PPE mutants. However, mwh cells differ by producing a larger number of hairs (typically three to four hairs) (Wong and Adler 1993). mwh mutations are epistatic to mutations in both the PCP and PPE genes and mwh is not required for the asymmetric accumulation of either PCP or PPE proteins (Usui et al. 1999; Strutt 2001; Adler et al. 2004; Strutt and Warrington 2008).The mwh gene was recently determined to encode a novel G protein binding–formin homology 3 (GBD-FH3) protein with a complex accumulation pattern in wing cells (Strutt and Warrington 2008; Yan et al. 2008). Prior to hair initiation Mwh accumulates along the proximal side of wing cells and during hair growth Mwh accumulates in the growing hair. Temperature-shift experiments with a temperature-sensitive allele provided evidence for two temporally separate mwh functions and it was proposed that the two accumulation patterns were associated with the two temporal functions (Yan et al. 2008). Here we show that the early proximal accumulation of Mwh requires the function of the PCP and PPE genes (a result also seen previously in Strutt and Warrington 2008), while the hair accumulation of Mwh is largely independent of these two groups of genes providing further genetic evidence for Mwh having two independent functions.How does the Mwh protein accumulate proximally? An obvious possibility is that Mwh interacts directly with one or more of the upstream proteins and in this way is recruited to the proximal side. The PPE proteins are strong candidates to interact directly with Mwh, as they function genetically in between the PCP gene and Mwh (Wong and Adler 1993). Consistent with this possibility we found that In and Mwh interacted in the yeast two-hybrid system and that these two proteins co-immunoprecipated from wing cells. This interaction was found not to be dependent on the function of the PCP genes consistent with the data from genetic studies that both in and mwh retain at least partial function in a fz mutant wing (Wong and Adler 1993). The hypothesis that Mwh is recruited to the proximal side by interacting with In predicts that these two proteins function in close proximity to one another. Consistent with these expectations we found that an In∷Mwh fusion protein provided both In and Mwh function.     

Epistatic Effects of the Protease/Chaperone HflB on Some Damaged Forms of the Escherichia coli Ammonium Channel AmtB

The Escherichia coli ammonium channel AmtB is a trimer in which each monomer carries a pore for substrate conduction and a cytoplasmic C-terminal extension of ∼25 residues. Deletion of the entire extension leaves the protein with intermediate activity, but some smaller lesions in this region completely inactivate AmtB, as do some lesions in its cytoplasmic loops. We here provide genetic evidence that inactivation depends on the essential protease HflB, which appears to cause inactivation not as a protease but as a chaperone. Selection for restored function of AmtB is a positive selection for loss of the ATPase/chaperone activity of HflB and reveals that the conditional lethal phenotype for hflB is cold sensitivity. Deletion of only a few residues from the C terminus of damaged AmtB proteins seems to prevent HflB from acting on them. Either yields the intermediate activity of a complete C-terminal deletion. HflB apparently “tacks” damaged AmtB tails to the adjacent monomers. Knowing that HflB has intervened is prerequisite to determining the functional basis for AmtB inactivation.Amt proteins concentrate the hydrated gas NH₄⁺ against a gradient and appear to be “active” channels (Andrade and Einsle 2007; Fong et al. 2007; Ludewig et al. 2007). Each monomer of the trimer carries a pore for substrate conduction and a C-terminal extension of variable length. The ordered C terminus of Escherichia coli AmtB is a long α-helix interrupted in the middle by a sharp kink, a fold very similar to that of the C-terminal region of Amt-1 from Archaeoglobus fulgidis (Andrade et al. 2005). The C terminus binds precisely to short cytoplasmic loop regions within the monomer to which it is covalently attached and also the adjacent monomer. It completes the cytoplasmic vestibule of the adjacent monomer to link the two (Conroy et al. 2007; Gruswitz et al. 2007). Deletion of the entire C terminus of E. coli AmtB yields a trimeric form of the protein with partial activity (Coutts et al. 2002; Severi et al. 2007): Uptake of [¹⁴C]methylammonium by a strain carrying AmtB^ΔC-term is between that of wild type and an amtB null strain.In organisms that are sensitive to the ammonium analog methylammonium, selection for resistance yields lesions in Amt proteins (Monahan et al. 2002; J. Hsu, W. B. Inwood and S. Kustu, unpublished results). These include some lesions that change residues in the C-terminal kink. Hence we introduced changes into the kink of the E. coli AmtB protein. All three that we tried—G393A, L394A, and the combination of the two—completely inactivated the protein, indicating that something had occurred beyond loss of function of the C terminus (Coutts et al. 2002; Severi et al. 2007). Likewise, we changed charged residues in the cytoplasmic loops of AmtB to alanine and this, too, inactivated the protein in several cases. By selecting for growth at NH₃ concentrations <50 nm, where unmediated diffusion is limiting, we isolated strains carrying a large number of mutations that suppressed the growth defect caused by these C-terminal and loop lesions. This work characterizes the intragenic suppressor mutations that affected the C terminus of AmtB, which were about a quarter of the total, and all of the extragenic suppressor mutations, which were ∼40%. It provides genetic evidence that the protease/chaperone HflB attempts to fold damaged C termini unsuccessfully and that this results in loss of AmtB activity. In the absence of intervention by HflB, the mutant C termini mimic a C-terminal delete.HflB (also called FtsH, which is unfortunately a misnomer because the hflB lesion was not responsible for the filamentation that was observed) is a membrane-bound protease that is the only essential ATP-dependent protease in E. coli (Ito and Akiyama 2005). It acts as a processive endopeptidase to release peptides of ∼20 residues. To digest inner-membrane proteins, it requires an N- or C-terminal cytoplasmic extension of about this length. HflB is divided into three regions: an N-terminal membrane-bound region containing two transmembrane segments separated by a large periplasmic loop (residues 1–143), an ATPase segment (AAA⁺ class; residues 144–398), and an unusual metalloprotease segment (residues 399–649) (Krzywda et al. 2002; Ito and Akiyama 2005; Bieniossek et al. 2006). Although the conditional lethal phenotype for hflB was long thought to be heat sensitivity, this has been questioned (Ogura et al. 1999). A deletion of hflB is tolerated in the presence of a suppressor mutation in fabZ that increases FabZ activity and restores the balance between phospholipid and lipopolysaccharide synthesis. The deletion is likewise tolerated in lpxA or lpxD backgrounds that decrease lipopolysacharide synthesis. HflB has been considered by some a charonin (Schumann 1999; Ito and Akiyama 2005), but there are few specific reports of its chaperone activity, and it is known to have difficulty unfolding proteins that are thermodynamically stable (Herman et al. 2003).     

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4′-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′-O-β-d-galactoside, resveratrol 4′-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.     

The Conserved miR-51 microRNA Family Is Redundantly Required for Embryonic Development and Pharynx Attachment in Caenorhabditis elegans

A major question about cytokinesis concerns the role of the septin proteins, which localize to the division site in all animal and fungal cells but are essential for cytokinesis only in some cell types. For example, in Schizosaccharomyces pombe, four septins localize to the division site, but deletion of the four genes produces only a modest delay in cell separation. To ask if the S. pombe septins function redundantly in cytokinesis, we conducted a synthetic-lethal screen in a septin-deficient strain and identified seven mutations. One mutation affects Cdc4, a myosin light chain that is an essential component of the cytokinetic actomyosin ring. Five others cause frequent cell lysis during cell separation and map to two loci. These mutations and their dosage suppressors define a signaling pathway (including Rho1 and a novel arrestin) for repairing cell-wall damage. The seventh mutation affects the poorly understood RNA-binding protein Scw1 and severely delays cell separation when combined either with a septin mutation or with a mutation affecting the septin-interacting, anillin-like protein Mid2, suggesting that Scw1 functions in a pathway parallel to that of the septins. Taken together, our results suggest that the S. pombe septins participate redundantly in one or more pathways that cooperate with the actomyosin ring during cytokinesis and that a septin defect causes septum defects that can be repaired effectively only when the cell-integrity pathway is intact.THE fission yeast Schizosaccharomyces pombe provides an outstanding model system for studies of cytokinesis (McCollum and Gould 2001; Balasubramanian et al. 2004; Pollard and Wu 2010). As in most animal cells, successful cytokinesis in S. pombe requires an actomyosin ring (AMR). The AMR begins to assemble at the G₂/M transition and involves the type II myosin heavy chains Myo2 and Myp2 and the light chains Cdc4 and Rlc1 (Wu et al. 2003). Myo2 and Cdc4 are essential for cytokinesis under all known conditions, Rlc1 is important at all temperatures but essential only at low temperatures, and Myp2 is essential only under stress conditions. As the AMR constricts, a septum of cell wall is formed between the daughter cells. The primary septum is sandwiched by secondary septa and subsequently digested to allow cell separation (Humbel et al. 2001; Sipiczki 2007). Because of the internal turgor pressure of the cells, the proper assembly and structural integrity of the septal layers are essential for cell survival.Septum formation involves the β-glucan synthases Bgs1/Cps1/Drc1, Bgs3, and Bgs4 (Ishiguro et al. 1997; Le Goff et al. 1999; Liu et al. 1999, 2002; Martín et al. 2003; Cortés et al. 2005) and the α-glucan synthase Ags1/Mok1 (Hochstenbach et al. 1998; Katayama et al. 1999). These synthases are regulated by the Rho GTPases Rho1 and Rho2 and the protein kinase C isoforms Pck1 and Pck2 (Arellano et al. 1996, 1997, 1999; Nakano et al. 1997; Hirata et al. 1998; Calonge et al. 2000; Sayers et al. 2000; Ma et al. 2006; Barba et al. 2008; García et al. 2009b). The Rho GTPases themselves appear to be regulated by both GTPase-activating proteins (GAPs) and guanine-nucleotide-exchange factors (GEFs) (Nakano et al. 2001; Calonge et al. 2003; Iwaki et al. 2003; Tajadura et al. 2004; Morrell-Falvey et al. 2005; Mutoh et al. 2005; García et al. 2006, 2009a,b). In addition, septum formation and AMR function appear to be interdependent. In the absence of a normal AMR, cells form aberrant septa and/or deposit septal materials at random locations, whereas a mutant defective in septum formation (bgs1) is also defective in AMR constriction (Gould and Simanis 1997; Le Goff et al. 1999; Liu et al. 1999, 2000). Both AMR constriction and septum formation also depend on the septation initiation network involving the small GTPase Spg1 (McCollum and Gould 2001; Krapp and Simanis 2008). Despite this considerable progress, many questions remain about the mechanisms and regulation of septum formation and its relationships to the function of the AMR.One major question concerns the role(s) of the septins. Proteins of this family are ubiquitous in fungal and animal cells and typically localize to the cell cortex, where they appear to serve as scaffolds and diffusion barriers for other proteins that participate in a wide variety of cellular processes (Longtine et al. 1996; Gladfelter et al. 2001; Hall et al. 2008; Caudron and Barral 2009). Despite the recent progress in elucidating the mechanisms of septin assembly (John et al. 2007; Sirajuddin et al. 2007; Bertin et al. 2008; McMurray and Thorner 2008), the details of septin function remain obscure. However, one prominent role of the septins and associated proteins is in cytokinesis. Septins concentrate at the division site in every cell type that has been examined, and in Saccharomyces cerevisiae (Hartwell 1971; Longtine et al. 1996; Lippincott et al. 2001; Dobbelaere and Barral 2004) and at least some Drosophila (Neufeld and Rubin 1994; Adam et al. 2000) and mammalian (Kinoshita et al. 1997; Surka et al. 2002) cell types, the septins are essential for cytokinesis. In S. cerevisiae, the septins are required for formation of the AMR (Bi et al. 1998; Lippincott and Li 1998). However, this cannot be their only role, because the AMR itself is not essential for cytokinesis in this organism (Bi et al. 1998; Korinek et al. 2000; Schmidt et al. 2002). Moreover, there is no evidence that the septins are necessary for AMR formation or function in any other organism. A further complication is that in some cell types, including most Caenorhabditis elegans cells (Nguyen et al. 2000; Maddox et al. 2007) and some Drosophila cells (Adam et al. 2000; Field et al. 2008), the septins do not appear to be essential for cytokinesis even though they localize to the division site.S. pombe has seven septins, four of which (Spn1, Spn2, Spn3, and Spn4) are expressed in vegetative cells and localize to the division site shortly before AMR constriction and septum formation (Longtine et al. 1996; Berlin et al. 2003; Tasto et al. 2003; Wu et al. 2003; An et al. 2004; Petit et al. 2005; Pan et al. 2007; Onishi et al. 2010). Spn1 and Spn4 appear to be the core members of the septin complex (An et al. 2004; McMurray and Thorner 2008), and mutants lacking either of these proteins do not assemble the others at the division site. Assembly of a normal septin ring also depends on the anillin-like protein Mid2, which colocalizes with the septins (Berlin et al. 2003; Tasto et al. 2003). Surprisingly, mutants lacking the septins are viable and form seemingly complete septa with approximately normal timing. These mutants do, however, display a variable delay in separation of the daughter cells, suggesting that the septins play some role(s) in the proper completion of the septum or in subsequent processes necessary for cell separation (Longtine et al. 1996; An et al. 2004; Martín-Cuadrado et al. 2005).It is possible that the septins localize to the division site and yet are nonessential for division in some cell types because their role is redundant with that of some other protein(s) or pathway(s). To explore this possibility in S. pombe, we screened for mutations that were lethal in combination with a lack of septins. The results suggest that the septins cooperate with the AMR during cytokinesis and that, in the absence of septin function, the septum is not formed properly, so that an intact system for recognizing and repairing cell-wall damage becomes critical for cell survival.     

Isolation and characterization of ribosomal ribonucleic acid

1. Ribosomal RNA has been prepared by extracting tissues with a phenol–cresol mixture, and ribosomal RNA can be selectively precipitated with m-cresol. No rapidly labelled RNA was associated with this material. 2. However, if RNA and DNA are extracted with 4-aminosalicylate and phenol–cresol mixture and the nucleic acids precipitated, DNA, glycogen and s-RNA (transfer RNA) can be extracted with 3m-sodium acetate and in this case rapidly labelled RNA remains associated with the ribosomal RNA. 3. The ribosomal RNA is stable in the presence of concentrated salt solution and, although the secondary structure is lost by heating at 70° in 10mm-sodium acetate, it can be re-formed in the presence of 200mm-sodium acetate. 4. The 28s and 18s components have been separated and their base compositions determined.     

Substrate specificity of the Bacillus licheniformis lyxose isomerase YdaE and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on d-lyxose, suggesting that the enzyme is d-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²⁺. The apparent K_m values for d-lyxose and d-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k_cat/K_m) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for d-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of d-lyxose and d-glucose from d-xylose and d-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM d-lyxose and d-mannose, 3.7 mM and 3.8 mM d-lyxose and d-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.     

Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22μmol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7–0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37°C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.     

Commentary on Urinary l-erythro-β-hydroxyasparagine: a novel serine racemase inhibitor and substrate of the Zn2+-dependent d-serine dehydratase

The analysis of the urine contents can be informative of physiological homoeostasis, and it has been speculated that the levels of urinary d-serine (d-ser) could inform about neurological and renal disorders. By analysing the levels of urinary d-ser using a d-ser dehydratase (DSD) enzyme, Ito et al. (Biosci. Rep.(2021) 41, BSR20210260) have described abundant levels of l-erythro-β-hydroxyasparagine (l-β-EHAsn), a non-proteogenic amino acid which is also a newly described substrate for DSD. The data presented support the endogenous production l-β-EHAsn, with its concentration significantly correlating with the concentration of creatinine in urine. Taken together, these results could raise speculations that l-β-EHAsn might have unexplored important biological roles. It has been demonstrated that l-β-EHAsn also inhibits serine racemase with K_i values (40 μM) similar to its concentration in urine (50 μM). Given that serine racemase is the enzyme involved in the synthesis of d-ser, and l-β-EHAsn is also a substrate for DSD, further investigations could verify if this amino acid would be involved in the metabolic regulation of pathways involving d-ser.     

The Power of the Methods for Detecting Interlocus Gene Conversion

Interlocus gene conversion can homogenize DNA sequences of duplicated regions with high homology. Such nonvertical events sometimes cause a misleading evolutionary interpretation of data when the effect of gene conversion is ignored. To avoid this problem, it is crucial to test the data for the presence of gene conversion. Here, we performed extensive simulations to compare four major methods to detect gene conversion. One might expect that the power increases with increase of the gene conversion rate. However, we found this is true for only two methods. For the other two, limited power is expected when gene conversion is too frequent. We suggest using multiple methods to minimize the chance of missing the footprint of gene conversion.INTERLOCUS (ectopic or nonallelic) gene conversion occurs between paralogous regions such that their DNA sequences are shuffled and homogenized (Petes and Hill 1988; Harris et al. 1993; Goldman and Lichten 1996). As a consequence, the DNA sequences of paralogous genes become similar (i.e., concerted evolution, Ohta 1980; Dover 1982; Arnheim 1983). This homogenizing effect of gene conversion sometimes causes problems in the inference of the evolutionary history of duplicated genes or multigene family. Common misleading inferences include an underestimation of the age of duplicated genes (Gao and Innan 2004; Teshima and Innan 2004). This is largely because the concept of the molecular clock is automatically incorporated in most software of phylogenetic analyses, and those software are frequently applied to multigene families without careful consideration of the potential effect of gene conversion.To understand the evolutionary roles of gene duplication, it is crucial to date each duplication event. To do this, we first need to know precisely the action of gene conversion among the gene family of interest. There have been a number of methods for detecting gene conversion, but their power has not been fully explored. Here, we systematically compare their performance by simulations to provide a guideline on which method works best under what condition. Our simulations show that some methods have a serious problem that causes a misleading interpretation: they do not detect any evidence for gene conversion when the gene conversion rate is too high. Thus, as is always true, lack of evidence is no evidence for absence, and we must be very careful about this effect when analyzing data with those tests, as is demonstrated below.There seem to be four major ideas behind the methods for detecting gene conversion, which are summarized below. A number of methods have been developed to detect interlocus gene conversion, and they belong to one of these four broad categories.

1. Incompatibility between an estimated gene tree and the true duplication history: Figure 1A illustrates a simple situation of a pair of duplicated genes, X and Y, that arose before the speciation event of species A and B. The upper tree of Figure 1A shows a tree representing the true history. When a gene tree is estimated from their DNA sequences, it should be consistent with the true tree when genes X and Y have accumulated mutations independently. Gene conversion potentially violates this relationship. When genes X and Y are subject to frequent gene conversion, the two paralogous genes in each species should be more closely related, resulting in a gene tree illustrated in the bottom tree in Figure 1A. Thus, incongruence between the real tree and an inferred gene tree can provide strong evidence for gene conversion (unless there is no lineage sorting or misinference of the gene tree).Open in a separate windowFigure 1.—Summary of the simulations in the two-species two-locus model. (A) Illustration of the model. (B–E) The power of the four approaches. The average gene conversion tract length (1/q) is assumed to be 100 bp. See Figure S1 for the results with 1/q = 1000 bp.It should be noted that a single gene conversion event usually transfers a short fragment. Consequently, it occasionally happens that incongruence is detected only in a part of the duplicated region. Thus, searching local regions of incongruence has been a well-recognized method for detecting nonvertical evolutionary events such as recombination, gene conversion, and horizontal gene transfer (Farris 1971; Brown et al. 1972), and some computational methods based on this idea have been developed (Balding et al. 1992).
2. Incompatibility of gene trees in different subregions: The idea of (i) can work even without knowing the real history. As mentioned above, incompatibility in the tree shape between different subregions can be evidence for local gene conversion because those subregions should have different histories of gene conversion (Sneath et al. 1975; Stephens 1985). A number of statistical algorithms incorporate this idea (e.g., Jakobsen et al. 1997; McGuire et al. 1997; Weiller 1998).
3. GENECONV: A local gene conversion also leaves its trace in the alignment of sequences. GENECONV is a software developed by Sawyer (1989) to detect such signatures (http://www.math.wustl.edu/∼sawyer/geneconv/). GENECONV looks at an alignment of multiple sequences in a pairwise manner and searches unusually long regions of high identity between the focal pair conditional on the pattern of variable sites in the other sequences, which are candidates of recent gene conversion (a similar idea is also seen in Sneath et al. 1975). The statistical significance is determined by random shuffling of variable sites in the alignment.
4. Shared polymorphism: Suppose polymorphism data are available in both of the duplicated genes. Then, with gene conversion, there could be polymorphisms shared by the two genes, which can be evidence for gene conversion (Innan 2003a). It should be noted that parallel mutations can create shared polymorphism even without gene conversion, but the chance should be very low when the point mutation rate is usually very low. Polymorphism data usually have tremendous amounts of information on very recent events and can be a powerful means to detect gene conversion (e.g., Stephens 1985; Betrán et al. 1997; Innan 2002).

In this study, we investigate and compare the performance of the methods based on these four ideas with simple settings. It should be noted that because our primary focus is on interlocus gene conversion, we ignore methods that can be used for detecting only allelic gene conversion, such as Fearnhead and Donnelly (2001), Hudson (2001), and Gay et al. (2007).