The DUB/USP17 deubiquitinating enzymes, a multigene family within a tandemly repeated sequence

Here we report the identification of 10 human, 1 murine, and 2 rat ORFs, all of which represent additional members of the DUB/USP17 family of deubiquitinating enzymes. In addition, we demonstrate that this family constitutes part of a tandemly repeated sequence conserved throughout humans, mice, and rats. Furthermore, upon examination of the known family members we have found that the multiple genes observed, in contrast to other gene families, have arisen due to the independent expansion of an ancestral sequence within each species. This premise is further strengthened by the observation that the murine and rat genes span two exons while their human counterparts have one. These observations, in conjunction with previous work demonstrating that the DUB/USP17's are cytokine inducible and that they regulate both cell growth and survival, suggest that the DUB/USP17's are a large highly conserved family of genes that may play an important role in controlling cell fate.

Intragenic SNP haplotypes associated with 84dup18 mutation in TNFRSF11A in four FEO pedigrees suggest three independent origins for this mutation.

Familial expansile osteolysis (FEO) is a rare disorder causing bone dysplasia. The clinical features of FEO include early-onset hearing loss, tooth destruction, and progressive lytic expansion within limb bones causing pain, fracture, and deformity. An 18-bp duplication in the first exon of the TNFRSF11A gene encoding RANK has been previously identified in four FEO pedigrees. Despite having the identical mutation, phenotypic variations among affected individuals of the same and different pedigrees were noted. Another 18-bp duplication, one base proximal to the duplication previously reported, was subsequently found in two unrelated FEO patients. Finally, mutations overlapping with the mutations found in the FEO pedigrees have been found in ESH and early-onset PDB pedigrees. An Iranian FEO pedigree that contains six affected individuals dispersed in three generations has previously been introduced; here, the clinical features of the proband are reported in greater detail, and the genetic defect of the pedigree is presented. Direct sequencing of the entire coding region and upstream and downstream noncoding regions of TNFRSF11A in her DNA revealed the same 18-bp duplication mutation as previously found in the four FEO pedigrees. Additionally, eight sequence variations as compared to the TNFRSF11A reference sequence were identified, and a haplotype linked to the mutation based on these variations was defined. Although the mutation in the Iranian and four of the previously described FEO pedigrees was the same, haplotypes based on the intragenic SNPs suggest that the mutations do not share a common descent.

A transposase-independent mechanism gives rise to precise excision of IS256 from insertion sites in Staphylococcus epidermidis.

The mobile element IS256 causes phase variation of biofilm formation in Staphylococcus epidermidis by insertion and precise excision from the icaADBC operon. Precise excision, i.e., removal of the target site duplications (TSDs) and restoration of the original DNA sequence, occurs rarely but independently of functional transposase. Instead, the integrity of the TSDs is crucial for precise excision. Excision increased significantly when the TSDs were brought into closer spatial proximity, suggesting that excision is a host-driven process that might involve most likely illegitimate recombination.

A Population-Based Study of IGF Axis Polymorphisms and the Esophageal Inflammation, Metaplasia, Adenocarcinoma Sequence

BACKGROUND & AIMS: Insulin-like growth factor (IGF) axis plays a key role in cell development, proliferation, and survival and is implicated in the etiology of several cancers. Few studies have examined the relationship between genetic variation of this axis and esophageal adenocarcinoma (EAC) or its precursors. METHODS: In a population-based case-control study, we investigated the association of common polymorphisms of IGF-1, IGF-2, IGF-1 receptor, IGF binding protein -3, growth hormones (GH) 1 and GH2, and GH receptor with reflux esophagitis (RE), Barrett esophagus (BE), and EAC. Two hundred and thirty RE, 224 BE, 227 EAC cases, and 260 controls were studied. Gene polymorphisms were identified using publicly available online resources; 102 IGF axis tag and putatively functional single-nucleotide polymorphisms (SNPs) were analyzed using MassARRAY iPLEX and Taqman assays. Results were analyzed using Haploview.
RESULTS: Three polymorphisms were disease-associated. IGF1 SNP rs6214 was associated with BE (adjusted P = .039). Using GG genotype as reference, odds ratio for BE in AA (wild-type) was 0.43 (95% confidence interval [CI], 0.24-0.75). GH receptor SNP rs6898743 was associated with EAC (adjusted P = .0112). With GG as reference, odds ratio for EAC in CC (wildtype) genotype was 0.42 (95% CI, 0.23-0.76). IGF1 (CA)(17) 185-bp allele was associated with RE (adjusted P = .0116). Using IGF1(non17) as reference, odds ratio for RE in IGF1(17) carriers was 7.29 (95% CI, 1.57-46.7).
CONCLUSIONS: In this study, 3 polymorphisms of IGF genes were associated with EAC or its precursors. These polymorphisms may be markers of disease risk; independent validation of our findings is required. These results suggest the IGF pathway is involved in EAC development.

The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluste

Background: The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A). Subsequently we have identified a number of human family members and shown that one of these (DUB-3) is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1.

Results: Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable betadefensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating.

Conclusions: The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related(1). These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae(2-5), closely related to the kelps(6,7) (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic(2) approaches to explore these and other(4,5) aspects of brown algal biology further.

The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases(*)

WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5'-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X(9)GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the beta-configuration, whereas the sugars bound to Und-PP are in the alpha-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases.

The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat

During O antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenylpyrophosphate (Und-P-P)-bound O antigen subunits occurs before their polymerization and ligation to the rest of the LPS molecule. Despite the general nature of the translocation process, putative O-antigen translocases display a low level of amino acid sequence similarity. In this work, we investigated whether complete O antigen subunits are required for translocation. We demonstrate that a single sugar, GlcNAc, can be incorporated to LPS of Escherichia coli K-12. This incorporation required the functions of two O antigen synthesis genes, wecA (UDP-GlcNAc:Und-P GlcNAc-1-P transferase) and wzx (O-antigen translocase). Complementation experiments with putative O-antigen translocases from E. coli O7 and Salmonella enterica indicated that translocation of O antigen subunits is independent of the chemical structure of the saccharide moiety. Furthermore, complementation with putative translocases involved in synthesis of exopolysaccharides demonstrated that these proteins could not participate in O antigen assembly. Our data indicate that recognition of a complete Und-P-P-bound O antigen subunit is not required for translocation and suggest a model for O antigen synthesis involving recognition of Und-P-P-linked sugars by a putative complex made of Wzx translocase and other proteins involved in the processing of O antigen.

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron-sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

Three motAB stator gene products in Bdellovibrio bacteriovorus contribute to motility of a single flagellum during predatory and prey-independent growth

The predatory bacterium Bdellovibrio bacteriovorus uses flagellar motility to locate regions rich in Gram-negative prey bacteria, colliding and attaching to prey and then ceasing flagellar motility. Prey are then invaded to form a "bdelloplast" in a type IV pilus-dependent process, and prey contents are digested, allowing Bdellovibrio growth and septation. After septation, Bdellovibrio flagellar motility resumes inside the prey bdelloplast prior to its lysis and escape of Bdellovibrio progeny. Bdellovibrio can also grow slowly outside prey as long flagellate host-independent (HI) cells, cultured on peptone-rich media. The B. bacteriovorus HD100 genome encodes three pairs of MotAB flagellar motor proteins, each of which could potentially form an inner membrane ion channel, interact with the FliG flagellar rotor ring, and produce flagellar rotation. In 2004, Flannagan and coworkers (R. S. Flannagan, M. A. Valvano, and S. F. Koval, Microbiology 150:649-656, 2004) used antisense RNA and green fluorescent protein (GFP) expression to downregulate a single Bdellovibrio motA gene and reported slowed release from the bdelloplast and altered motility of the progeny. Here we inactivated each pair of motAB genes and found that each pair contributes to motility, both predatorily, inside the bdelloplast and during HI growth; however, each pair was dispensable, and deletion of no pair abolished motility totally. Driving-ion studies with phenamil, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and different pH and sodium conditions indicated that all Mot pairs are proton driven, although the sequence similarities of each Mot pair suggests that some may originate from halophilic species. Thus, Bdellovibrio is a "dedicated motorist," retaining and expressing three pairs of mot genes.

Independent genomewide screens identify the tumor suppressor VTRNA2-1 as a human epiallele responsive to periconceptional environment

Background: Interindividual epigenetic variation that occurs systemically must be established prior to gastrulation in the very early embryo and, because it is systemic, can be assessed in easily biopsiable tissues. We employ two independent genome-wide approaches to search for such variants.

Results: First, we screen for metastable epialleles by performing genomewide bisulfite sequencing in peripheral blood lymphocyte (PBL) and hair follicle DNA from two Caucasian adults. Second, we conduct a genomewide screen for genomic regions at which PBL DNA methylation is affected by season of conception in rural Gambia. Remarkably, both approaches identify the genomically imprinted VTRNA2-1 as a top environmentally responsive epiallele. We demonstrate systemic and stochastic interindividual variation in DNA methylation at the VTRNA2-1 differentially methylated region in healthy Caucasian and Asian adults and show, in rural Gambians, that periconceptional environment affects offspring VTRNA2-1 epigenotype, which is stable over at least 10 years. This unbiased screen also identifies over 100 additional candidate metastable epialleles, and shows that these are associated with cis genomic features including transposable elements.

Conclusions: The non-coding VTRNA2-1 transcript (also called nc886) is a putative tumor suppressor and modulator of innate immunity. Thus, these data indicating environmentally induced loss of imprinting at VTRNA2-1 constitute a plausible causal pathway linking early embryonic environment, epigenetic alteration, and human disease. More broadly, the list of candidate metastable epialleles provides a resource for future studies of epigenetic variation and human disease.

Exome sequencing of prostate cancer supports the hypothesis of independent tumour origins

BACKGROUND: Prostate cancer (PCa) is a clinically and pathologically heterogeneous disease. The rapid development of sequencing technology has the potential to deliver new biomarkers with emphasis on aggressive disease and to revolutionise personalised cancer treatment. However, a prostate harbouring cancer commonly contains multiple separate tumour foci, with the potential to aggravate tumour sampling. The level of intraprostatic tumour heterogeneity remains to be determined.

OBJECTIVE: To determine the level of intraprostatic tumour heterogeneity through genome-wide, high-resolution profiling of multiple tumour samples from the same individual.

DESIGN, SETTINGS, AND PARTICIPANTS: Multiple tumour samples were obtained from four individuals following radical prostatectomy. One individual (SWE-1) contained >70% cancer cells in all tumour samples, whereas the other three (SWE-2 to SWE-4) required the use of laser capture microdissection for tumour cell enrichment. Subsequently, DNA was extracted from all tissue samples, and exome sequencing was performed. All tumour foci of SWE-1 were also profiled using a high-resolution array for the identification of copy number alterations (CNA).

OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Shared somatic high-frequency single nucleotide variants (SNV) and CNAs were used to infer the level of intraprostatic tumour heterogeneity.

RESULTS AND LIMITATIONS: No high-frequency mutations, common for the three tumour samples of SWE-1, were identified. Ten randomly chosen positions were validated with Sanger sequencing in all foci, which verified the exome data. The high level of intraprostatic heterogeneity was consistent in all individuals. In total, three out of four individuals harboured tumours without an apparent common somatic denominator. Although we cannot exclude the presence of common structural rearrangements, a high-density array was used for the detection of deletions and amplifications in SWE-1, which agreed with the exome data.

CONCLUSIONS: We present evidence for the presence of somatically independent tumours within the same prostate. This finding will have implications for personalised cancer treatment and biomarker discovery.

Sequence variation in the CHAT locus shows no association with late-onset Alzheimer's disease

There is substantial evidence for a susceptibility gene for late-onset Alzheimer's disease (AD) on chromosome 10. One of the characteristic features of AD is the degeneration and dysfunction of the cholinergic system. The genes encoding choline acetyltransferase (ChAT) and its vesicular transporter (VAChT), CHAT and SLC18A3 respectively, map to the linked region of chromosome 10 and are therefore both positional and obvious functional candidate genes for late-onset AD. We have screened both genes for sequence variants and investigated each for association with late-onset AD in up to 500 late-onset AD cases and 500 control DNAs collected in the UK. We detected a total of 17 sequence variants. Of these, 14 were in CHAT, comprising three non-synonymous variants (D7N in the S exon, A120T in exon 5 and L243F in exon 8), one synonymous change (H547H), nine single-nucleotide polymorphisms in intronic, untranslated or promoter regions, and a variable number of tandem repeats in intron 7. Three non-coding SNPs were detected in SLC18A3. None demonstrated any reproducible association with late-onset AD in our samples. Levels of linkage disequilibrium were generally low across the CHAT locus but two of the coding variants, D7N and A120T, proved to be in complete linkage disequilibrium.