A Genetic Screen for Suppressors of a Mutated 5′ Splice Site Identifies Factors Associated With Later Steps of Spliceosome Assembly

Many alleles of human disease genes have mutations within splicing consensus sequences that activate cryptic splice sites. In Caenorhabditis elegans, the unc-73(e936) allele has a G-to-U mutation at the first base of the intron downstream of exon 15, which results in an uncoordinated phenotype. This mutation triggers cryptic splicing at the −1 and +23 positions and retains some residual splicing at the mutated wild-type (wt) position. We previously demonstrated that a mutation in sup-39, a U1 snRNA gene, suppresses e936 by increasing splicing at the wt splice site. We report here the results of a suppressor screen in which we identify three proteins that function in cryptic splice site choice. Loss-of-function mutations in the nonessential splicing factor smu-2 suppress e936 uncoordination through changes in splicing. SMU-2 binds SMU-1, and smu-1(RNAi) also leads to suppression of e936. A dominant mutation in the conserved C-terminal domain of the C. elegans homolog of the human tri-snRNP 27K protein, which we have named SNRP-27, suppresses e936 uncoordination through changes in splicing. We propose that SMU-2, SMU-1, and SNRP-27 contribute to the fidelity of splice site choice after the initial identification of 5′ splice sites by U1 snRNP.PRE-mRNA splicing takes place in a large ribonucleoprotein complex called the spliceosome (Burge et al. 1999). Components of this splicing machinery assemble at conserved signal sequences within the pre-mRNA. The 5′ splice site consensus sequence M₋₃A₋₂G₋₁ | G₊₁U₊₂R₊₃A₊₄G₊₅U₊₆ and the 3′ splice site consensus sequence Y₋₃A₋₂G₋₁ | R₊₁ (M is either A or C; R is a purine, and Y is a pyrimidine) define the limits of the intron. Base-pairing interactions between the 5′ end of the U1 snRNA and the 5′ splice site consensus sequence occur early in spliceosome assembly. It is the nearly invariable GU dinucleotide at the first two positions of the 5′ end of the intron that defines the beginning of the intron. The 5′ consensus sequence is essential but insufficient for splice site selection, as 5′ splice sites with weaker consensus matches may require additional determinants for proper activation (Sanford et al. 2005).Mutations that disrupt the 5′ consensus splice signal can lead to genetic disease in humans (Nelson and Green 1990; Cohen et al. 1994). Approximately 15% of point mutations that cause genetic diseases affect pre-mRNA splicing consensus sequences (Krawczak et al. 1992). For some specific disease genes, as many as 50% of the known heritable alleles alter splicing (Teraoka et al. 1999; Ars et al. 2000; Roca et al. 2003; Pagenstecher et al. 2006). Among all the positions of the 5′ splice site consensus sequence, the highest proportion of human disease mutations occur at the +1G position (Buratti et al. 2007). The fidelity of pre-mRNA splice site choice is largely disrupted by this defect, since this mutation causes splicing at this site to be either abolished or outcompeted by the activation of nearby cryptic 5′ splice sites (Nelson and Green 1990; Cohen et al. 1994). Cryptic splice sites are used only when the wild-type splice donor is disrupted by mutation, as they tend to have very weak splice donor consensus sequences outside of a 5′-GU dinucleotide that defines the beginning of the intron (Roca et al. 2003). Suppression of mutations to the 5′ splice site consensus sequence in vivo has been achieved through the expression of U1 snRNAs containing compensatory base substitutions (Zhuang and Weiner 1986); however, suppression of mutations to the +1 position of the intron using reverse genetic approaches has not been successful (Newman et al. 1985; Nelson and Green 1990; Cohen et al. 1994).We have used a specific allele of the Caenorhabditis elegans unc-73 gene, e936, which contains a G-to-U mutation at the first nucleotide of intron 16 (Steven et al. 1998), as a model for studying cryptic splice site choice (Roller et al. 2000; Zahler et al. 2004). unc-73 encodes a RAC guanine nucleotide exchange factor that is expressed in neurons and is important for axon guidance (Steven et al. 1998). The e936 allele induces the use of three different cryptic 5′ splice sites (Figure 1A). Two of these 5′ splice sites, located at the −1 and +23 positions, define introns beginning with GU. The third 5′ splice site used is at the mutated wild-type (wt) position and is referred to as “wt” since splicing at this site still produces wild-type unc-73 mRNA and protein, even though the intron begins with UU (Roller et al. 2000). Use of either the −1 or the +23 cryptic site causes a shift in the reading frame and loss of gene function. In e936 animals, 90% of the stable messages of unc-73 are out-of-frame, yet the phenotype is not as severe as for other alleles in this gene. This indicates that the 10% of steady-state messages that are in frame have some functional role.Open in a separate windowFigure 1.—(A) Diagram of the unc-73 gene between exons 15 and 16. The positions of the −1 and +23 cryptic 5′ splice sites are indicated by arrows. The intronic e936 (+1G → U) point mutation is highlighted. (B) γ-³²P-labeled RT–PCR results across the cryptic splicing region of unc-73(e936) for different strains. Lanes 1, 2, and 3 are loaded with RT–PCR reactions from wild type (N2), unc-73(e936);sup-39(je5), and unc-73(e936) RNA, respectively. The lines carrying the suppressor alleles and e936 follow in lanes 4–10 as indicated. (C) The unc-73 genomic sequence from exon 15 (uppercase letters) and intron 15 (lowercase letters). The locations of the az23 and e936 mutational substitutions are indicated below. The position of the −9 cryptic splice donor activated in e936az23 is indicated by an arrow above.In a previous genetic screen for extragenic suppressors of e936 movement defects, Way and colleagues identified sup-39 (Run et al. 1996). It was subsequently shown that mutations in sup-39 alter cryptic splice site choice of e936 (Roller et al. 2000). sup-39 encodes a U1 snRNA gene with a compensatory mutation at the position that normally base pairs with the +1G. This allows sup-39 to base pair with an intron with a +1U (Zahler et al. 2004). This dominant suppressor increases usage of the mutated splice site and improves the fraction of in-frame messages from e936 from 10 to 33%, with a dramatic improvement in coordination. A similar mutant U1 snRNA suppressor with a different compensatory substitution, sup-6(st19), was found to suppress the intronic +1G to A transition of unc-13(e309) to allow for splicing at the mutated wild-type site, even though the intron begins with AU instead of GU (Zahler et al. 2004).We are interested in identifying additional factors that play a role in cryptic 5′ splice site choice. To do this, we took advantage of unc-73(e936), in which modest increases in the use of the wt splice site lead to dramatic increases in coordination, as a sensitive screen for changes in cryptic splice site choice. In this article we report that the proteins SMU-1 and SMU-2, which are nonessential factors previously shown to have a role in alternative splicing (Spartz et al. 2004), have a role in selection of cryptic 5′ splice sites. We also report the identification of a new dominant suppressor of cryptic splicing, snrp-27, which encodes a C. elegans homolog of the human tri-snRNP 27K protein.     

Recharacterization of the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104)

Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-β-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.     

Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli

Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (K _m = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (K _m = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.     

Spp382p Interacts with Multiple Yeast Splicing Factors,Including Possible Regulators of Prp43 DExD/H-Box Protein Function

Prp43p catalyzes essential steps in pre-mRNA splicing and rRNA biogenesis. In splicing, Spp382p stimulates the Prp43p helicase to dissociate the postcatalytic spliceosome and, in some way, to maintain the integrity of the spliceosome assembly. Here we present a dosage interference assay to identify Spp382p-interacting factors by screening for genes that when overexpressed specifically inhibit the growth of a conditional lethal prp38-1 spliceosome assembly mutant in the spp382-1 suppressor background. Identified, among others, are genes encoding the established splicing factors Prp8p, Prp9p, Prp11p, Prp39p, and Yhc1p and two poorly characterized proteins with possible links to splicing, Sqs1p and Cwc23p. Sqs1p copurifies with Prp43p and is shown to bind Prp43p and Spp382p in the two-hybrid assay. Overexpression of Sqs1p blocks pre-mRNA splicing and inhibits Prp43p-dependent steps in rRNA processing. Increased Prp43p levels buffer Sqs1p cytotoxicity, providing strong evidence that the Prp43p DExD/H-box protein is a target of Sqs1p. Cwc23p is the only known yeast splicing factor with a DnaJ motif characteristic of Hsp40-like chaperones. We show that similar to SPP382, CWC23 activity is critical for efficient pre-mRNA splicing and intron metabolism yet, surprisingly, this activity does not require the canonical DnaJ/Hsp40 motif. These and related data establish the value of this dosage interference assay for finding genes that alter cellular splicing and define Sqs1p and Cwc23p as prospective modulators of Spp382p-stimuated Prp43p function.EIGHT phylogenetically conserved DExD/H-box proteins act at discrete steps to regulate the assembly, activation, and dissociation of the splicing apparatus (reviewed in Brow 2002; Konarska and Query 2005; Linder 2006). How these RNA-dependent ATPases are temporally and functionally regulated remains poorly understood. One putative regulator, the 83-kDa Spp382/Ntr1 protein (henceforth referred to by the Saccharomyces Genome Database standard name, Spp382p), was discovered in a screen for mutants capable of suppressing defects in yeast spliceosome assembly (Pandit et al. 2006). While spp382 null alleles are lethal, partial loss of function suppresses mutations in several other splicing factors, including the genes encoding the essential spliceosomal proteins Prp8p and Prp38p. Spp382p is a spliceosomal protein that binds the Prp43p DExD/H-box protein to promote efficient dissociation of spliceosomal factors after completion of splicing in vitro (Tsai et al. 2005). Some but not all spp382 mutants also accumulate the excised intron product of splicing in vivo, ostensibly due to protection of the intron within a hyperstabilized spliceosome (Pandit et al. 2006; Tanaka et al. 2007). The suppression of spliceosome assembly defects by spp382 mutation is proposed to occur by impairing Spp382p-stimulated dissociation of kinetically impaired or otherwise inefficient spliceosomes by Prp43p (Pandit et al. 2006). Consistent with this hypothesis, prp43 mutations also suppress spliceosome assembly defects in a manner that, within limits, is inversely proportional to the residual ATPase activity of Prp43p (Pandit et al. 2006). In this light, it is possible that the Spp382 and Prp43 proteins are components of the “discard pathway” for spliceosome dissociation predicted by the kinetic proofreading model of DExD/H-box protein function (Burgess et al. 1990; Konarska and Query 2005).Recently, several groups made the surprising observation that the Prp43p splicing factor is also required for ribosome biogenesis. Mutations in PRP43 inhibit 35S pre-rRNA cleavage and limit downstream steps in this processing pathway (Lebaron et al. 2005; Combs et al. 2006; Leeds et al. 2006). Prp43p is ≥10-fold more abundant than the splicing-restricted DExD/H-box proteins (e.g., Brr2p, Prp2p, Prp5, Prp16, Prp22p, and Prp28p; see Ghaemmaghami et al. 2003) and, consistent with dual function in splicing and rRNA processing, nuclear Prp43p is enriched in the nucleolus (Huh et al. 2003). Furthermore, proteins and small RNAs acting exclusively in splicing or in rRNA biogenesis copurify with Prp43p, supporting its direct contribution to both processes (Ho et al. 2002; Lebaron et al. 2005; Combs et al. 2006; Gavin et al. 2006; Krogan et al. 2006; Leeds et al. 2006). While it is not certain how Prp43p is partitioned within the cell, its association with Spp382p appears critical for recruitment to the postcatalytic spliceosome and for stimulation of the intrinsic Prp43p helicase activity (Tsai et al. 2005; Boon et al. 2006; Pandit et al. 2006; Tanaka et al. 2007). A structurally related protein, Pxr1p, interacts with Prp43p (Lebaron et al. 2005) and is required for efficient rRNA processing (Guglielmi and Werner 2002). Pxr1p may serve a parallel role for Prp43p recruitment and activation within the rRNA processing apparatus but direct evidence for such function is lacking.Here we describe a genetic approach to identify factors that interact with SPP382 and function in spliceosome dynamics. Specifically, we describe a dosage interference assay to find genes that when overexpressed inhibit the growth of a yeast strain in which the temperature-sensitive prp38-1 spliceosome assembly mutation (Xie et al. 1998) is suppressed by the spp382-1 suppressor allele (Pandit et al. 2006). We identify multiple GAL1-effector genes that preferentially inhibit growth of the prp38-1 spp382-1 double mutant compared with either single-mutant host or a wild-type yeast strain. Consistent with the goal of this screen, most genes cause splicing inhibition with galactose induction.Among the recovered genes are SQS1 and CWC23, which encode proteins that purify from yeast in multisubunit protein complexes containing Prp43p but have unknown functions (Lebaron et al. 2005; Pandit et al. 2006). Sqs1p is a nonessential 87-kDa protein that, similar to the Prp43p interacting proteins Pxr1p and Spp382p, possesses the glycine-rich G-patch motif common to a subset of RNA binding proteins (Aravind and Koonin 1999). CWC23 encodes a 33-kDa protein with a canonical DnaJ motif characteristic of Hsp40-like activators of Hsp70 chaperones (Walsh et al. 2004; Vos et al. 2008). Cwc23p interacts with Spp382p in the two-hybrid assay and by affinity selection (Pandit et al. 2006). The genetic and biochemical results presented here establish Sqs1p and Cwc23p as Spp382p-interacting proteins with contributions to RNA processing distinct from what might be expected on the basis of their protein motif characteristics.     

Physiology and Substrate Specificity of Two Closely Related Amino Acid Transporters,SerP1 and SerP2, of Lactococcus lactis

The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports l-serine, l-threonine, and l-cysteine with high affinity. Affinity constants (K_m) are in the 20 to 40 μM range. SerP2 is a dl-alanine/dl-serine/glycine transporter. The preferred substrate appears to be dl-alanine for which the affinities were found to be 38 and 20 μM for the d and l isomers, respectively. The common substrate l-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 μM, respectively. Growth experiments demonstrate that SerP1 is the main l-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substrates l-alanine and glycine. SerP2 plays an adverse role for the cell by being solely responsible for the uptake of toxic d-serine. The main function of SerP2 is in cell wall biosynthesis through the uptake of d-alanine, an essential precursor in peptidoglycan synthesis. SerP2 has overlapping substrate specificity and shares 42% sequence identity with CycA of Escherichia coli, a transporter whose involvement in peptidoglycan synthesis is well established. No evidence was obtained for a role of SerP1 and SerP2 in the excretion of excess amino acids during growth of L. lactis on protein/peptide-rich media.     

Estimating the Parameters of Selection on Nonsynonymous Mutations in Drosophila pseudoobscura and D. miranda

We present the results of surveys of diversity in sets of >40 X-linked and autosomal loci in samples from natural populations of Drosophila miranda and D. pseudoobscura, together with their sequence divergence from D. affinis. Mean silent site diversity in D. miranda is approximately one-quarter of that in D. pseudoobscura; mean X-linked silent diversity is about three-quarters of that for the autosomes in both species. Estimates of the distribution of selection coefficients against heterozygous, deleterious nonsynonymous mutations from two different methods suggest a wide distribution, with coefficients of variation greater than one, and with the average segregating amino acid mutation being subject to only very weak selection. Only a small fraction of new amino acid mutations behave as effectively neutral, however. A large fraction of amino acid differences between D. pseudoobscura and D. affinis appear to have been fixed by positive natural selection, using three different methods of estimation; estimates between D. miranda and D. affinis are more equivocal. Sources of bias in the estimates, especially those arising from selection on synonymous mutations and from the choice of genes, are discussed and corrections for these applied. Overall, the results show that both purifying selection and positive selection on nonsynonymous mutations are pervasive.SURVEYS of DNA sequence diversity and divergence are shedding light on a number of questions in evolutionary genetics (for recent reviews, see Akey 2009; Sella et al. 2009). Two of the most important questions of this kind concern the distribution of selection coefficients against deleterious mutations affecting protein sequences and the proportion of amino acid sequence differences between related species that have been fixed by positive selection. Several different methods have been proposed for studying each of these questions, using different features of data on polymorphism and divergence at nonsynonymous and silent sites.For example, the parameters of the distribution of selection coefficients against deleterious amino acid mutations have been estimated by contrasting the numbers of nonsynonymous and silent within-species polymorphisms and fixed differences between species (Sawyer and Hartl 1992; Bustamante et al. 2002; Piganeau and Eyre-Walker 2003; Sawyer et al. 2007); by fitting the frequency spectra of nonsynonymous and silent variants to models of selection, mutation, and drift (Akashi 1999; Eyre-Walker et al. 2006; Keightley and Eyre-Walker 2007; Kryukov et al. 2007; Boyko et al. 2008; Eyre-Walker and Keightley 2009); or by comparing levels of nonsynonymous and silent diversities between species with different population sizes (Loewe and Charlesworth 2006; Loewe et al. 2006). The results of these different approaches generally agree in suggesting that there is a wide distribution of selection coefficients against nonsynonymous mutations and that the mean selection coefficient against heterozygous carriers of such mutations is very small. The results imply that a typical individual from a human population carries several hundred weakly deleterious mutations (Eyre-Walker et al. 2006; Kryukov et al. 2007; Boyko et al. 2008); for a typical Drosophila population, with its much higher level of variability, the number is probably an order of magnitude greater (Loewe et al. 2006; Keightley and Eyre-Walker 2007).The presence of this large load of slightly deleterious mutations in human and natural populations, most of which are held at low frequencies by natural selection, has many implications. From the point of view of understanding human genetic disease, it means that we have to face the likelihood that susceptibility to a disease can be influenced by variants at many loci, each with small effects (Kryukov et al. 2007). The pervasive presence of deleterious mutations throughout the genome contributes to inbreeding depression (Charlesworth and Willis 2009) and may mean that the effective population size is reduced by background selection effects, even in regions of the genome with normal levels of genetic recombination (Loewe and Charlesworth 2007). Their presence may contribute so strongly to Hill–Robertson effects (Hill and Robertson 1966; Felsenstein 1974) that they cause severely reduced levels of diversity and adaptation in low-recombination regions of the genome (Charlesworth et al. 2010) and create a selective advantage to maintaining nonzero levels of recombination (Keightley and Otto 2006; Charlesworth et al. 2010). In addition, having an estimate of the distribution of selection coefficients against deleterious nonsynonymous mutations allows their contribution to between-species divergence to be predicted, providing a way of estimating the fraction of fixed nonsynonymous differences caused by positive selection (Loewe et al. 2006; Boyko et al. 2008; Eyre-Walker and Keightley 2009).It is thus important to collect data that shed light on the properties of selection against nonsynonymous mutations in a wide range of systems and also to compare the results from different methods of estimation, since they are subject to different sources of difficulty and biases. In a previous study, we proposed the use of a comparison between two related species with different effective population sizes for this purpose (Loewe and Charlesworth 2006; Loewe et al. 2006), using Drosophila miranda and D. pseudoobscura as material. These are well suited for this type of study, as they are closely related, live together in similar habitats, and yet have very different levels of silent nucleotide diversity, indicating different effective population sizes (N_e). This study was hampered by our inability to compare the same set of loci across the two species and by the small number of loci that could be used. We here present the results of a much larger study of DNA variation at X-linked and autosomal loci for these two species, using D. affinis as a basis for estimating divergence. We compare the results, applying the method of Loewe et al. (2006) with that of Eyre-Walker and Keightley (2009) for estimating the distribution of deleterious selection coefficients and with McDonald–Kreitman test-based methods for estimating the proportion of nonsynonymous differences fixed by positive selection. While broadly confirming the conclusions from earlier studies, we note some possible sources of bias and describe methods for minimizing their effects.     

On the inference of complex phylogenetic networks by Markov Chain Monte-Carlo

For various species, high quality sequences and complete genomes are nowadays available for many individuals. This makes data analysis challenging, as methods need not only to be accurate, but also time efficient given the tremendous amount of data to process. In this article, we introduce an efficient method to infer the evolutionary history of individuals under the multispecies coalescent model in networks (MSNC). Phylogenetic networks are an extension of phylogenetic trees that can contain reticulate nodes, which allow to model complex biological events such as horizontal gene transfer, hybridization and introgression. We present a novel way to compute the likelihood of biallelic markers sampled along genomes whose evolution involved such events. This likelihood computation is at the heart of a Bayesian network inference method called SnappNet, as it extends the Snapp method inferring evolutionary trees under the multispecies coalescent model, to networks. SnappNet is available as a package of the well-known beast 2 software.Recently, the MCMC_BiMarkers method, implemented in PhyloNet, also extended Snapp to networks. Both methods take biallelic markers as input, rely on the same model of evolution and sample networks in a Bayesian framework, though using different methods for computing priors. However, SnappNet relies on algorithms that are exponentially more time-efficient on non-trivial networks. Using simulations, we compare performances of SnappNet and MCMC_BiMarkers. We show that both methods enjoy similar abilities to recover simple networks, but SnappNet is more accurate than MCMC_BiMarkers on more complex network scenarios. Also, on complex networks, SnappNet is found to be extremely faster than MCMC_BiMarkers in terms of time required for the likelihood computation. We finally illustrate SnappNet performances on a rice data set. SnappNet infers a scenario that is consistent with previous results and provides additional understanding of rice evolution.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.