AID and Apobec3G haphazard deamination and mutational diversity

Activation-induced deoxycytidine deaminase (AID) and Apobec 3G (Apo3G) cause mutational diversity by initiating mutations on regions of single-stranded (ss) DNA. Expressed in B cells, AID deaminates C → U in actively transcribed immunoglobulin (Ig) variable and switch regions to initiate the somatic hypermutation (SHM) and class switch recombination (CSR) that are essential for antibody diversity. Apo3G expressed in T cells catalyzes C deaminations on reverse transcribed cDNA causing HIV-1 retroviral inactivation. When operating properly, AID- and Apo3G-initiated mutations boost human fitness. Yet, both enzymes are potentially powerful somatic cell “mutators”. Loss of regulated expression and proper genome targeting can cause human cancer. Here, we review well-established biological roles of AID and Apo3G. We provide a synopsis of AID partnering proteins during SHM and CSR, and describe how an Apo2 crystal structure provides “surrogate” insight for AID and Apo3G biochemical behavior. However, large gaps remain in our understanding of how dC deaminases search ssDNA to identify trinucleotide motifs to deaminate. We discuss two recent methods to analyze ssDNA scanning and deamination. Apo3G scanning and deamination is visualized in real-time using single-molecule FRET, and AID deamination efficiencies are determined with a random walk analysis. AID and Apo3G encounter many candidate deamination sites while scanning ssDNA. Generating mutational diversity is a principal aim of AID and an important ancillary property of Apo3G. Success seems likely to involve hit and miss deamination motif targeting, biased strongly toward miss.     

New frontiers of primary antibody deficiencies

Primary antibody deficiencies (PAD) form the largest group of inherited disorders of the immune system. They are characterized by a marked reduction or absence of serum immunoglobulins (Ig) due to disturbed B cell differentiation and by a poor response to vaccination. PAD can be divided into agammaglobulinemia, Ig class switch recombination deficiencies, and idiopathic hypogammaglobulinemia. Over the past 20 years, defects have been identified in 18 different genes, but in many PAD patients the underlying gene defects have not been found. Diagnosis of known PAD and discovery of new PAD is important for good patient care. In this review, we present the effects of genetic defects in the context of normal B cell differentiation, and we discuss how new technical developments can support understanding and discovering new genetic defects in PAD.     

Common evolution of waprin and kunitz-like toxin families in Australian venomous snakes

The venoms of Australian snakes contain a myriad of pharmacologically active toxin components. This study describes the identification and comparative analysis of two distinct toxin families, the kunitztype serine protease inhibitors and waprins, and demonstrates a previously unknown evolutionary link between the two. Multiple cDNA and full-length gene isoforms were cloned and shown to be composed of three exons separated by two introns. A high degree of identity was observed solely within the first exon which coded for the propeptide sequence and its cleavage site, and indicates that each toxin family has arisen from a gene duplication event followed by diversification only within the portion of the gene coding for the functional toxin. It is proposed that while the mechanism of toxin secretion is highly conserved, diversification of mature toxin sequences allows for the existence of multiple protein isoforms in the venom to adapt to variations within the prey environment.     

Na+ binding to meizothrombin desF1

Meizothrombin is the physiologically active intermediate generated by a single cleavage of prothrombin at R320 to separate the A and B chains. Recent evidence has suggested that meizothrombin, like thrombin, is a Na(+)-activated enzyme. In this study we present the first X-ray crystal structure of human meizothrombin desF1 solved in the presence of the active site inhibitor PPACK at 2.1 A resolution. The structure reveals a Na(+) binding site whose architecture is practically identical to that of human thrombin. Stopped-flow measurements of Na(+) binding to meizothrombin desF1 document a slow phase of fluorescence change with a k(obs) decreasing hyperbolically with increasing [Na(+)], consistent with the existence of three conformations in equilibrium, E*, E and E:Na(+), as for human thrombin. Evidence that meizothrombin exists in multiple conformations provides valuable new information for studies of the mechanism of prothrombin activation.     

Sex determination in mammals — Before and after the evolution of SRY

Therian mammals (marsupials and placentals) have an XX female: XY male sex chromosome system, which is homologous to autosomes in other vertebrates. The testis-determining gene, SRY, is conserved on the Y throughout therians, but is absent in other vertebrates, suggesting that the mammal system evolved about 310 million years ago (MYA). However, recent work on the basal monotreme mammals has completely changed our conception of how and when this change occurred. Platypus and echidna lack SRY, and the therian X and Y are represented by autosomes, implying that SRY evolved in therians after their divergence from monotremes only 166 MYA. Clues to the ancestral mechanism usurped by SRY in therians are provided by the monotremes, whose sex chromosomes are homologous to the ZW of birds. This suggests that the therian X and Y, and the SRY gene, evolved from an ancient bird-like sex chromosome system which predates the divergence of mammals and reptiles 310 MYA. Received 4 March 2008; received after revision 22 April 2008; accepted 3 June 2008     

A novel member of the Rhomboid family, RHBDD1, regulates BIK-mediated apoptosis

Rhomboid family members are widely conserved and found in all three kingdoms of life. They are serine proteases and serve important regulatory functions. In the present study, a novel gene highly expressed in the testis, RHBDD1, is shown to be a new member of the Rhomboid family, participating in the cleavage of BIK, a proapoptotic member of the Bcl-2 family. The RHBDD1-involved proteolytic modification is upstream of the BIK protein degradation pathway. Mutagenesis studies show that the amino acid residues glycine142 and serine144 of RHBDD1 are crucial for its activity in cleaving BIK at a site located in the transmembrane region. Overexpression or knock-down of RHBDD1 in HEK 293T cells can reduce or enhance BIK-mediated apoptosis, respectively. The present findings suggest that, by acting as a serine protease, RHBDD1 modulates BIK-mediated apoptotic activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 31 July 2008; received after revision 16 September 2008; accepted 19 September 2008     

Gene conversion in the chicken immunoglobulin locus: A paradigm of homologous recombination in higher eukaryotes

Gene conversion was first defined in yeast as a type of homologous recombination in which the donor sequence does not change. In chicken B cells, gene conversion builds the antigen receptor repertoire by introducing sequence diversity into the immunoglobulin genes. Immunoglobulin gene conversion continues at high frequency in an avian leukosis virus induced chicken B cell line. This cell line can be modified by homologous integration of transfected DNA constructs offering a model system for studying gene conversion in higher eukaryotes. In search for genes which might participate in chicken immunoglobulin gene conversion, we have identified chicken counterparts of the yeastRAD51, RAD52, andRAD54 genes. Disruption and overexpression of these genes in the chicken B cell line may clarify their role in gene conversion and gene targeting.     

Hydroxylation of demethoxy-Q6 constitutes a control point in yeast coenzyme Q6 biosynthesis

Coenzyme Q is a lipid molecule required for respiration and antioxidant protection. Q biosynthesis in Saccharomyces cerevisiae requires nine proteins (Coq1p–Coq9p). We demonstrate in this study that Q levels are modulated during growth by its conversion from demethoxy-Q (DMQ), a late intermediate. Similar conversion was produced when cells were subjected to oxidative stress conditions. Changes in Q₆/DMQ₆ ratio were accompanied by changes in COQ7 gene mRNA levels encoding the protein responsible for the DMQ hydroxylation, the penultimate step in Q biosynthesis pathway. Yeast coq null mutant failed to accumulate any Q late biosynthetic intermediate. However, in coq7 mutants the addition of exogenous Q produces the DMQ synthesis. Similar effect was produced by over-expressing ABC1/COQ8. These results support the existence of a biosynthetic complex that allows the DMQ₆ accumulation and suggest that Coq7p is a control point for the Q biosynthesis regulation in yeast. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 04 September 2008; received after revision 22 October 2008; accepted 23 October 2008     

The genomic basis of the Williams – Beuren syndrome

The Williams-Beuren syndrome is a genomic disorder (prevalence: 1/7,500 to 1/20,000), caused by a hemizygous contiguous gene deletion on chromosome 7q11.23. Typical symptoms comprise supravalvular aortic stenosis, mental retardation, overfriendliness and visuospatial impairment. The common deletion sizes range of 1.5–1.8 mega base pairs (Mb), encompassing app. 28 genes. For a few genes, a genotype-phenotype correlation has been established. The best-explored gene within this region is the elastin gene; its haploinsufficiency causes arterial stenosis. The region of the Williams-Beuren syndrome consists of a single copy gene region (~1.2 Mb) flanked by repetitive sequences – Low Copy Repeats (LCR). The deletions arise as a consequence of misalignment of these repetitive sequences during meiosis and a following unequal crossing over due to high similarity of LCRs. This review presents an overview of the Williams-Beuren syndrome region considering the genomic assembly, chromosomal rearrangements and their mechanisms (i.e. deletions, duplications, inversions) and evolutionary and historical aspects. Received 11 July 2008; received after revision 15 October 2008; accepted 16 October 2008     

Generating spinal motor neuron diversity: a long quest for neuronal identity

Understanding how thousands of different neuronal types are generated in the CNS constitutes a major challenge for developmental neurobiologists and is a prerequisite before considering cell or gene therapies of nervous lesions or pathologies. During embryonic development, spinal motor neurons (MNs) segregate into distinct subpopulations that display specific characteristics and properties including molecular identity, migration pattern, allocation to specific motor columns, and innervation of defined target. Because of the facility to correlate these different characteristics, the diversification of spinal MNs has become the model of choice for studying the molecular and cellular mechanisms underlying the generation of multiple neuronal populations in the developing CNS. Therefore, how spinal motor neuron subpopulations are produced during development has been extensively studied during the last two decades. In this review article, we will provide a comprehensive overview of the genetic and molecular mechanisms that contribute to the diversification of spinal MNs.     

Effects of ulapualide A and synthetic macrolide analogues on actin dynamics and gene regulation

Several marine macrolide toxins act as potent and specific actin-severing molecules. Recent elucidation of their stereochemistries and modes of interaction with actin has allowed the syntheses of bioactive analogues. Here we used synthetic analogues in a structure-function analysis of ulapualide A, a trisoxazole-based macrolide. Ulapualide A harboured potent actin-depolymerising activity both in cells and in vitro. Its synthetic diastereoisomer was three orders of magnitude less active than the natural toxin and synthetic macrolide fragments lacked actin-capping/ severing activity altogether. Modulation of serum response factor (SRF)-dependent gene expression, as described for other actin-binding toxins, was also examined. Specific changes in response to ulapualide A were not observed, primarily due to its profound effects on cytoskeletal integrity and cell adhesion. Several synthetic fragments of ulapualide A also had no effect on SRF-dependent gene expression. However, inhibition was observed with a molecule corresponding to the extended aliphatic side chain of halichondramide, a structurally related macrolide. These findings indicate that side-chain derivatives of trisoxazole-based macrolides may serve to uncouple gene-regulatory events from actin dynamics. E. Vincent and J. Saxton: These two authors contributed equally Received 27 September 2006; received after revision 30 November 2006; accepted 8 January 2007     

Luciferase a light source for the silica-based optical waveguides (spicules) in the demosponge Suberites domuncula

Two classes of sponges (animal phylum Porifera) possess a siliceous skeleton which is composed of spicules. Studying the optical fiber-mechanical properties of large spicules from hexactinellid sponges (> 5 cm) it was demonstrated that they are effective light-collecting optical fibers. Here, we report that the demosponge Suberites domuncula is provided with a biosensor system composed of the (organic) light producing luciferase and the (inorganic) light transducing silica spicules. The light transmission feature of these smaller spicules (200 μm) has been demonstrated and the ability of sponge tissue to generate light has been proven. Screening for a luciferase gene in S. domuncula was successful; the recombinant luciferase was prepared and shown to be bioactive. The luciferase protein is abundantly present in the close neighborhood of the spicules. The expression of the luciferase gene is under the control of light. Received 14 August 2008; received after revision 09 November 2008; accepted 26 November 2008