DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency.

DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS). Some of the mutations identified in the patients directly disrupt the ligase domain while others impair the interaction between DNA ligase IV and Xrcc-4. Cell lines from the patients show pronounced radiosensitivity. Unlike NBS cell lines, they show normal cell cycle checkpoint responses but impaired DNA double-strand break rejoining. An unexpected V(D)J recombination phenotype is observed involving a small decrease in rejoining frequency coupled with elevated imprecision at signal junctions.     

Envelope structure of Synechococcus sp. WH8113, a nonflagellated swimming cyanobacterium

Background  

Many bacteria swim by rotating helical flagellar filaments [1]. Waterbury et al. [15] discovered an exception, strains of the cyanobacterium Synechococcus that swim without flagella or visible changes in shape. Other species of cyanobacteria glide on surfaces [2,7]. The hypothesis that Synechococcus might swim using traveling surface waves [6,13] prompted this investigation.     

Evolutionary relationships among "jakobid" flagellates as indicated by alpha- and beta-tubulin phylogenies

Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists. They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakobiformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J. libera and R. americana, the two other jakobids, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads. A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J. libera-R. americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies. In alpha- tubulin trees, J. incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on the phylogenetic method employed. Tubulin gene phylogenies were in general agreement with mitochondrial gene phylogenies and ultrastructural data in indicating that the "jakobids" may be polyphyletic. Relationships with the putatively deep-branching amitochondriate diplomonads remain uncertain.     

Microbial diversity of hydrothermal sediments in the Guaymas Basin: evidence for anaerobic methanotrophic communities

Microbial communities in hydrothermally active sediments of the Guaymas Basin (Gulf of California, Mexico) were studied by using 16S rRNA sequencing and carbon isotopic analysis of archaeal and bacterial lipids. The Guaymas sediments harbored uncultured euryarchaeota of two distinct phylogenetic lineages within the anaerobic methane oxidation 1 (ANME-1) group, ANME-1a and ANME-1b, and of the ANME-2c lineage within the Methanosarcinales, both previously assigned to the methanotrophic archaea. The archaeal lipids in the Guaymas Basin sediments included archaeol, diagnostic for nonthermophilic euryarchaeota, and sn-2-hydroxyarchaeol, with the latter compound being particularly abundant in cultured members of the Methanosarcinales. The concentrations of these compounds were among the highest observed so far in studies of methane seep environments. The delta-(13)C values of these lipids (delta-(13)C = -89 to -58 per thousand) indicate an origin from anaerobic methanotrophic archaea. This molecular-isotopic signature was found not only in samples that yielded predominantly ANME-2 clones but also in samples that yielded exclusively ANME-1 clones. ANME-1 archaea therefore remain strong candidates for mediation of the anaerobic oxidation of methane. Based on 16S rRNA data, the Guaymas sediments harbor phylogenetically diverse bacterial populations, which show considerable overlap with bacterial populations of geothermal habitats and natural or anthropogenic hydrocarbon-rich sites. Consistent with earlier observations, our combined evidence from bacterial phylogeny and molecular-isotopic data indicates an important role of some novel deeply branching bacteria in anaerobic methanotrophy. Anaerobic methane oxidation likely represents a significant and widely occurring process in the trophic ecology of methane-rich hydrothermal vents. This study stresses a high diversity among communities capable of anaerobic oxidation of methane.     

The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.     

Mutations in the NHEJ Component XRCC4 Cause Primordial Dwarfism

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation.     

Protein <Emphasis Type="Italic">N</Emphasis>-glycosylation,protein folding,and protein quality control

Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.     

<Emphasis Type="Italic">Phylo-mLogo</Emphasis>: an interactive and hierarchical multiple-logo visualization tool for alignment of many sequences

Background  

When aligning several hundreds or thousands of sequences, such as epidemic virus sequences or homologous/orthologous sequences of some big gene families, to reconstruct the epidemiological history or their phylogenies, how to analyze and visualize the alignment results of many sequences has become a new challenge for computational biologists. Although there are several tools available for visualization of very long sequence alignments, few of them are applicable to the alignments of many sequences.