Immunoglobulin light-chain genes in the rhesus macaque II: lambda light-chain germline sequences for subgroups IGLV1, IGLV2, IGLV3, IGLV4 and IGLV5

The combined processes of immunoglobulin (IG) gene rearrangement and somatic hypermutation allow for the creation of an extremely diverse antibody repertoire. Knowledge of the germline sequence of the IG genes is required so that hypermutation and the affinity matured humoral response can be properly studied. Variable region genes can be arranged into subgroups; in humans, there are 11 IGLV subgroups and 6 IGKV subgroups. The rhesus macaque (Macaca mulatta) is a relevant non-human primate model for human immunological systems. A number of macaque IGHV, IGHD and IGHJ genes have already been reported. We have also previously reported a number of macaque IGKV genes. Here we report the isolation of new macaque IGLV genes by polymerase chain reaction amplification from macaque genomic DNA using primers based on the human sequences. Nine IGLV1, 10 IGLV2, 21 IGLV3, 5 IGLV4 and 7 IGLV5 germline genes for the macaque were found, the open-reading frames of which exhibit high homology to their human counterparts (>89.3, >88.6, >89.0, >94.7 and >87.1%, respectively). Electronic supplementary material Supplementary material is available in the online version of this article at and accessible for authorised users. W.A. Howard and J.M. Bible contributed equally to this work.     

The center of the center of marine shore fish biodiversity: the Philippine Islands

Synopsis Multiple datasets show global maxima of marine biodiversity in the Indo–Malay–Philippines archipelago (IMPA). Analysis of distribution data for 2983 species reveals a pattern of richness on a finer scale and identifies a peak of marine biodiversity in the central Philippine Islands and a secondary peak between peninsular Malaysia and Sumatra. This pattern is repeated in diverse habitat and higher taxa classes, most rigorously for marine shore fishes, supporting geohistorical hypotheses as the most general unifying explanations. Specific predictions based on area of overlap, area of accumulation, and area of refuge hypotheses suggest that present day eastern Indonesia, or Wallacea, should be the center of marine biodiversity. Processes suggested by these three hypotheses contribute to the diversity in this region and are also a likely explanation for the secondary center of diversity. Our study indicates, however, that there is a higher concentration of species per unit area in the Philippines than anywhere in Indonesia, including Wallacea. The Philippine center of diversity is consistent with hypotheses that this area experienced numerous vicariant and island integration events and these hypotheses warrant further testing. Special attention to marine conservation efforts in the Philippines is justified because of the identification of it as an epicenter of biodiversity and evolution.     

A bayesian analysis of metazoan mitochondrial genome arrangements

Genome arrangements are a potentially powerful source of information to infer evolutionary relationships among distantly related taxa. Mitochondrial genome arrangements may be especially informative about metazoan evolutionary relationships because (1) nearly all animals have the same set of definitively homologous mitochondrial genes, (2) mitochondrial genome rearrangement events are rare relative to changes in sequences, and (3) the number of possible mitochondrial genome arrangements is huge, making convergent evolution of genome arrangements appear highly unlikely. In previous studies, phylogenetic evidence in genome arrangement data is nearly always used in a qualitative fashion-the support in favor of clades with similar or identical genome arrangements is considered to be quite strong, but is not quantified. The purpose of this article is to quantify the uncertainty among the relationships of metazoan phyla on the basis of mitochondrial genome arrangements while incorporating prior knowledge of the monophyly of various groups from other sources. The work we present here differs from our previous work in the statistics literature in that (1) we incorporate prior information on classifications of metazoans at the phylum level, (2) we describe several advances in our computational approach, and (3) we analyze a much larger data set (87 taxa) that consists of each unique, complete mitochondrial genome arrangement with a full complement of 37 genes that were present in the NCBI (National Center for Biotechnology Information) database at a recent date. In addition, we analyze a subset of 28 of these 87 taxa for which the non-tRNA mitochondrial genomes are unique where the assumption of our inversion-only model of rearrangement is more plausible. We present summaries of Bayesian posterior distributions of tree topology on the basis of these two data sets.     

Binding-protein expression is subject to temporal,developmental and stress-induced regulation in terminally differentiated soybean organs

Binding protein (BiP) is a widely distributed and highly conserved endoplasmic-reticulum luminal protein that has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of misfolded polypeptides. Analysis of cDNA sequences and genomic blots indicates that soybeans (Glycine max L. Merr.) possess a small gene family encoding BiP. The deduced sequence of BiP is very similar to that of other plant BiPs. We have examined the expression of BiP in several different terminally differentiated soybean organs including leaves, pods and seed cotyledons. Expression of BiP mRNA increases during leaf expansion while levels of BiP protein decrease. Leaf BiP mRNA is subject to temporal control, exhibiting a large difference in expression in a few hours between dusk and night. The expression of BiP mRNA varies in direct correlation with accumulation of seed storage proteins. The hybridization suggests that maturing-seed BiP is likely to be a different isoform from vegetative BiPs. Levels of BiP protein in maturing seeds vary with BiP mRNA. High levels of BiP mRNA are detected after 3 d of seedling growth. Little change in either BiP mRNA or protein levels was detected in maturing soybean pods, although BiP-protein levels decrease in fully mature pods. Persistent wounding of leaves by whiteflies induces massive overexpression of BiP mRNA while only slightly increasing BiP-protein levels. In contrast single-event puncture wounding only slightly induces additional BiP expression above the temporal variations. These observations indicate that BiP is not constitutively expressed in terminally differentiated plant organs. Expression of BiP is highest during the developmental stages of leaves, pods and seeds when their constituent cells are producing seed or vegetative storage proteins, and appears to be subject to complex regulation, including developmental, temporal and wounding.The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.Abbreviations BiP binding protein The sequences reported in this paper have been submitted to Gen-Bank and are identified with the accession numbers BiP-A (U08384), BiP-B (U08383), BiP-C (U08382) and -1,3 glucanase (U08405).     

Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone¹ H^N, ¹⁵N,¹³ C and¹³ C and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.     

Infanticide in Propithecus diadema edwardsi: An Evaluation of the Sexual Selection Hypothesis

Infanticide might be described as a reproductive strategy employed by anthropoid primate males when they immigrate into new groups. But infanticide has rarely been observed in wild prosimian primates. For the Malagasy lemurs this may reflect one or more of the following: strict breeding seasons; relative monomorphism in canine tooth and body size; small group sizes; male–female dominance relations; and male–female dyads within groups. We addressed the following questions: Do prosimian males commit infanticide in circumstances similar to those in which anthropoids do? and Is there any reproductive advantage for a highly seasonal breeder to commit infanticide? To help answer these questions, we describe the death of a 24-hr-old infant male Propithecus diadema edwardsi from wounds received during a fight between his mother, her adult daughter, and a newly immigrant male. Interbirth intervals between surviving offspring are 2 years for Propithecus diadema edwardsi; therefore, a male could dramatically shorten the time between reproductive windows by killing an infant. Whether this tactic would be favored by sexual selection cannot be addressed until more information has been collected on the length of interbirth interval due to infanticide relative to that of infant death by other causes; how social factors such as stability of breeding relationships affect long-term male reproductive success; how effective female counterstrategies are to prevent infanticide and/or whether they choose to mate with males that commit infanticide; and how often males that kill infants subsequently sire infants, particularly in groups that contain a resident male.     

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22^phox, creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H₂O₂ production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22^phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O₂⁻ generation. In contrast, Nox4 released H₂O₂ and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.The family of NADPH oxidases consists of seven members termed Nox/Duox that differ in their tissue expression profiles, modes of activation, reactive oxygen species (ROS) outputs, and physiological functions. Understanding their distinguishing features is a prerequisite for rational inhibitor design and thus targeted intervention in ROS-mediated pathophysiologies (4). The coexpression of different Nox isoforms, each with potentially distinct functional profiles, in the same cell type necessitates a more discriminating approach than application of pan-Nox inhibitors. Detailed structure-function studies are necessary to identify unique regions and their impact with respect to catalytic function or localization of the enzyme. All Nox/Duox enzymes share a Nox backbone with six predicted transmembrane domains and an intracellular carboxyl-terminal domain which harbors FAD and NADPH binding sites. Nox5 and Duox1/2 enzymes contain additional structural elements such as amino terminal EF-hand motifs, a hallmark of their regulation by the intracellular calcium concentration (13, 30).The founding member of the NADPH oxidase family, the phagocyte oxidase, consists of membrane-bound Nox2 in a complex with the smaller subunit p22^phox (3). Heterodimerization of these two proteins is required for maturation and translocation of the enzyme complex to the plasma membrane or to intracellular vesicles. The Nox family members Nox1, Nox3, and Nox4 follow this paradigm (1, 14, 21, 25, 31). Heterodimer formation and association of the Nox/p22^phox complex at particular cellular membranes is essential for catalytic activity, i.e., for ROS generation. Nox2, and to a lesser degree Nox1 and Nox3, remain dormant under resting conditions and rely on stimulus-dependent translocation and assembly of oxidase components such as p47^phox and p67^phox, or NoxO1 and NoxA1 in the case of Nox1 and Nox3 (16). These steps, together with activation and translocation of the GTPase Rac, ultimately lead to the assembled, catalytically active oxidase and to ROS generation.Nox4 differs from the usual theme of multimeric assembly of active NADPH oxidases found in Nox1 to Nox3 (21, 22, 28, 32). Constitutive H₂O₂ production by Nox4 localized at perinuclear vesicles has been reported (1, 21, 28). Since NADPH oxidases catalyze the one-electron reduction of molecular oxygen to superoxide anion, the current dogma suggests that Nox4 generates intracellular superoxide. The superoxide produced will then dismutate rapidly to H₂O₂, diffusing from the cell into the extracellular milieu. Cytosolic proteins, which regulate the activity of Nox1 to Nox3 by binding to the carboxyl-terminal domains of Nox1 to Nox3, seem to be irrelevant for Nox4 function. The membrane-bound subunit p22^phox is to date the only known protein associated with Nox1 to Nox4. Heterodimerization, translocation, and enzymatic function of these oxidases require p22^phox. Recent structure-function analyses of complexes between Nox2 or Nox4 and the subunit p22^phox documented specific regions and amino acid residues in p22^phox necessary for complex formation and oxidase activity (35, 37). Interestingly, a p22^phox mutant (p22^phox Y121H) is capable of distinguishing between Nox1 to Nox3 and Nox4 by forming a functional complex only with Nox4, further suggesting unique structural features in Nox4 (35).In this study, we expand structure-function analysis of the oxidase complex by comparing Nox4/Nox2 chimeric enzymes with respect to NADPH oxidase activity, type of reactive oxygen species produced, requirement for additional oxidase components, and detailed subcellular localization.     

Genomic Content of Neisseria Species

The physical properties of most bacterial genomes are largely unexplored. We have previously demonstrated that the strict human pathogen Neisseria gonorrhoeae is polyploid, carrying an average of three chromosome copies per cell and only maintaining one pair of replication forks per chromosome (D. M. Tobiason and H. S. Seifert, PLos Biol. 4:1069-1078, 2006). We are following up this initial report to test several predictions of the polyploidy model of gonococcal chromosome organization. We demonstrate that the N. gonorrhoeae chromosomes exist solely as monomers and not covalently linked dimers, and in agreement with the monomer status, we show that distinct nucleoid regions can be detected by electron microscopy. Two different approaches to isolate heterozygous N. gonorrhoeae resulted in the formation of merodiploids, showing that even with more than one chromosome copy, these bacteria are genetically haploid. We show that the closely related bacterium Neisseria meningitidis is also polyploid, while the commensal organism Neisseria lactamica maintains chromosomes in single copy. We conclude that the pathogenic Neisseria strains are homozygous diploids.Bacteria are unicellular organisms that exhibit a multitude of shapes and sizes and exist in a wide range of environments. Despite the extreme diversity of capabilities and physiology evidenced by different bacterial species, most bacteria are assumed to conform to the enteric model of genomic organization, chromosomal replication, and genomic segregation during cell division exemplified by Escherichia coli. In contradiction to this limited view of bacterial genome biology, some bacterial species have their genome divided between multiple DNA elements (10), and some possess linear chromosomes (2, 19). A few bacterial species have been reported to carry multiple genome copies per cell (members of the genera Azotobacter, Borrelia, Buchnera, Deinococcus, Neisseria, and Epulopiscium), with copy number estimates ranging from two copies to thousands of copies per cell (1, 7, 17, 23, 25, 26, 34, 35, 39, 46). The exact number of genomes per cell has not been determined for most of these organisms, and the mechanisms for organizing polyploid genomes and segregating them during cell division remain to be determined. An exception is Deinococcus radiodurans, which has been shown to possess four complete chromosomes during exponential growth and up to 16 genomes within the stationary phase. The polyploid genomes of D. radiodurans have been proposed to assemble into a toroidal mass in the cell (29), but the validity of this finding has been questioned (11, 13, 49). There are few obvious commonalities between these polyploid organisms, except that some Neisseria, Deinococcus, and Borrelia species utilize homologous recombination to mediate specialized processes essential for the survival of these species. In addition, members of the Azotobacter, Buchnera, and Epulopiscium genera are obligate symbionts that do not possess a free-living stage, but the reasons why obligate symbionts would possess polyploid chromosomes are unknown.Neisseria gonorrhoeae and Neisseria meningitidis are the two pathogenic members of the Neisseria genus. N. gonorrhoeae is the sole causative agent of the disease gonorrhea, and N. meningitidis is the most common cause of bacterial meningitis in adolescents and young adults. One attribute that these human-specific pathogens use to coexist and evolve within humans lies in their capacity to antigenically vary and phase vary several outer membrane structures, including pili, Opa proteins, and the lipooligosaccharide (LOS) (12, 21). Variation of the Opa and LOS antigens is mediated by polynucleotide repeat variation that modulates expression of biosynthetic genes (40, 48). These changes in polynucleotide repeat sequences are mediated through slipped-strand mispairing that occurs during normal DNA replication and therefore would not obviously benefit from polyploidy. In contrast, pilin antigenic variation is a RecA-mediated gene conversion event (27), which could be aided by having two copies of all the recombining pilin loci within a single cell to facilitate the nonreciprocal transfer of pilin sequences. Therefore, we postulated that the presence of multiple genome copies per gonococcal cell may be required to facilitate these high-frequency gene conversion events. Analysis of gonococcal genome content by flow cytometry and fluorescent microscopy indicated that there existed greater than one genome equivalent of gonococcal DNA content per cell (46). Additionally, quantitative real-time PCR and genome microarray analysis measured a marker frequency pinpointing a single DNA replication event per round of cell division. On average, each coccal unit had three genome copies per cell, and a population of cells with a single genome equivalent per cell was never observed, even under conditions of slower growth. These observations predicted a model for gonococcal replication (Fig. ​(Fig.1)1) in which each coccal unit has a minimum of two chromosomes that replicate in unison to produce four chromosomes prior to cell division and the conclusion that this species is diploid.Open in a separate windowFIG. 1.Model of gonococcal DNA replication and chromosome segregation in a monococcus. The gonococcal chromosome is indicated by a dotted line. An antibiotic resistance (Ab^R) marker recombined into a gonococcal chromosome (solid line). At time zero minutes, DNA replication begins, and after 35 min, DNA replication is complete. Gonococcal cell division occurs after 60 min. In scenario I, homozygous chromosomes are segregated together. In scenario II, heterozygous bacteria are produced.Though the analysis of the genomic content has only been reported for the gonococcus, the genus Neisseria encompasses a number of pathogenic and commensal bacteria. N. meningitidis is the leading cause of bacterial meningitis worldwide and is asymptomatically carried in the human nasopharynx (47). Most of the Neisseria species are commensal organisms that inhabit the nasopharynx and rarely cause disease (24). The most extensively studied commensal Neisseria species, N. lactamica, shares extensive homology with the pathogenic Neisseriae species and also predominately resides in the human nasopharynx (31). Only the pathogenic neisseriae, N. gonorrhoeae and N. meningitidis, have been shown to undergo pilin antigenic variation (43). Since the polyploid nature of N. gonorrhoeae has been proposed to be required for pilin antigenic variation, N. meningitidis may also have multiple genome copies per cell. The genomic copy number of other Neisseria species and the putative relationship between genomic content and pathogenesis remain to be determined.In this work, we tested several predictions or models resulting from the observation that the gonococcus is polyploid. We confirmed that the chromosomes exist as separate molecules and show that the gonococcal nucleoids reside in discrete cellular regions. We confirm that these bacteria are genetically haploid, suggesting that chromosomal segregation mechanisms ensure a homozygous population. Finally, we show that the other pathogenic Neisseria species, N. meningitidis, is also polyploid, while a commensal Neisseria species, N. lactamica, is not. These studies show that polyploidy is correlated with Neisseria pathogenesis and suggest that this property has evolved to allow diploid chromosomes while maintaining the haploid status of these obligate human pathogens.     

Functional Characterization of the Conserved “GLK” Motif in Mitochondrial Porin from Neurospora crassa

Mitochondrial porin facilitates the diffusion of small hydrophilic molecules across the mitochondrial outer membrane. Despite low sequence similarity among porins from different species, a glycine-leucine-lysine (GLK) motif is conserved in mitochondrial and Neisseria porins. To investigate the possible roles of these conserved residues, including their hypothesized participation in ATP binding by the protein, we replaced the lysine residue of the GLK motif of Neurospora crassa porin with glutamic acid through site-directed mutagenesis of the corresponding gene. Although the pores formed by this protein have size and gating characteristics similar to those of the wild-type protein, the channels formed by GLEporin are less anion selective than the wild-type pores. The GLEporin retains the ability to be cross linked to [-³²P]ATP, indicating that the GLK sequence is not essential for ATP binding. Furthermore, the pores formed by both GLEporin and the wild-type protein become more cation selective in the presence of ATP. Taken together, these results support structural models that place the GLK motif in a part of the ion-selective -barrel that is not directly involved in ATP binding.