Genome-wide analysis reveals DNA methylation markers that vary with both age and obesity

The combination of the obesity epidemic and an aging population presents growing challenges for the healthcare system. Obesity and aging are major risk factors for a diverse number of diseases and it is of importance to understand their interaction and the underlying molecular mechanisms. Herein the authors examined the methylation levels of 27578 CpG sites in 46 samples from adult peripheral blood. The effect of obesity and aging was ascertained with general linear models. More than one hundred probes were correlated to aging, nine of which belonged to the KEGG group map04080. Additionally, 10 CpG sites had diverse methylation profiles in obese and lean individuals, one of which was the telomerase catalytic subunit (TERT). In eight of ten cases the methylation change was reverted between obese and lean individuals. One region proved to be differentially methylated with obesity (LINC00304) independent of age. This study provides evidence that obesity influences age driven epigenetic changes, which provides a molecular link between aging and obesity. This link and the identified markers may prove to be valuable biomarkers for the understanding of the molecular basis of aging, obesity and associated diseases.     

Genome-wide analysis of re-replication reveals inhibitory controls that target multiple stages of replication initiation

DNA replication must be tightly controlled during each cell cycle to prevent unscheduled replication and ensure proper genome maintenance. The currently known controls that prevent re-replication act redundantly to inhibit pre-replicative complex (pre-RC) assembly outside of the G1-phase of the cell cycle. The yeast Saccharomyces cerevisiae has been a useful model organism to study how eukaryotic cells prevent replication origins from reinitiating during a single cell cycle. Using a re-replication-sensitive strain and DNA microarrays, we map sites across the S. cerevisiae genome that are re-replicated as well as sites of pre-RC formation during re-replication. Only a fraction of the genome is re-replicated by a subset of origins, some of which are capable of multiple reinitiation events. Translocation experiments demonstrate that origin-proximal sequences are sufficient to predispose an origin to re-replication. Origins that reinitiate are largely limited to those that can recruit Mcm2-7 under re-replicating conditions; however, the formation of a pre-RC is not sufficient for reinitiation. Our findings allow us to categorize origins with respect to their propensity to reinitiate and demonstrate that pre-RC formation is not the only target for the mechanisms that prevent genomic re-replication.     

Genome-wide association analysis reveals putative Alzheimer's disease susceptibility loci in addition to APOE

Alzheimer's disease (AD) is a genetically complex and heterogeneous disorder. To date four genes have been established to either cause early-onset autosomal-dominant AD (APP, PSEN1, and PSEN2(1-4)) or to increase susceptibility for late-onset AD (APOE5). However, the heritability of late-onset AD is as high as 80%, (6) and much of the phenotypic variance remains unexplained to date. We performed a genome-wide association (GWA) analysis using 484,522 single-nucleotide polymorphisms (SNPs) on a large (1,376 samples from 410 families) sample of AD families of self-reported European descent. We identified five SNPs showing either significant or marginally significant genome-wide association with a multivariate phenotype combining affection status and onset age. One of these signals (p = 5.7 x 10(-14)) was elicited by SNP rs4420638 and probably reflects APOE-epsilon4, which maps 11 kb proximal (r2 = 0.78). The other four signals were tested in three additional independent AD family samples composed of nearly 2700 individuals from almost 900 families. Two of these SNPs showed significant association in the replication samples (combined p values 0.007 and 0.00002). The SNP (rs11159647, on chromosome 14q31) with the strongest association signal also showed evidence of association with the same allele in GWA data generated in an independent sample of approximately 1,400 AD cases and controls (p = 0.04). Although the precise identity of the underlying locus(i) remains elusive, our study provides compelling evidence for the existence of at least one previously undescribed AD gene that, like APOE-epsilon4, primarily acts as a modifier of onset age.     

Prediction and analysis of paralogous proteins in Trichomonas vaginalis genome

Trichomonas vaginalis causes trichomoniasis, second most sexually transmitted disease. The genome sequence draft of T. vaginalis was published by The Institute of Genomic Research reveals an abnormally large genome size of 160 Mb. It was speculated that a significant portion of the proteome contains paralogous proteins. The present study was aimed at identification and analysis of the paralogous proteins. The all against all search approach is used to identify the paralogous proteins. The dataset of proteins was retrieved from TIGR and TrichDB FTP server. The BLAST-P program performed all against all database searches against the protein database of Trichomonas vaginalis available at NCBI genome database. In the present study about 50,000 proteins were searched where 2,700 proteins were found to be paralogous under the rigid selection criteria. The Pfam database search has identified significant number of paralogous proteins which were further categorized among different 1496 paralogous protein in pfam families, 1027 paralogous protein contains domain, 60 proteins were having different repeats and 1092 paralogous protein sequences of clans. Such identification and functional annotation of paralogous proteins will also help in removing paralogous proteins from possible drug targets in future. Presence of huge number of paralogous proteins across wide range of gene families and domains may be one of the possible mechanisms involved in the T. vaginalis genome expansion and evolution.     

Genome-wide association analysis reveals cryptic alleles as an important factor in heterosis for fatness in chicken F2 population

Genome-wide association studies have become possible in the chicken because of the recent availability of the complete genome sequence, a polymorphism map and high-density single nucleotide polymorphism (SNP) genotyping platforms. We used these tools to study the genetic basis of a very high level of heterosis that was previously observed for fatness in two F₂ populations established by crossing one outbred broiler (meat-type) sire with dams from two unrelated, highly inbred, light-bodied lines (Fayoumi and Leghorn). In each F₂ population, selective genotyping was carried out using phenotypically extreme males for abdominal fat percentage (AF) and about 3000 SNPs. Single-point association analysis of about 500 informative SNPs per cross showed significant association ( P  < 0.01) of 15 and 24 markers with AF in the Broiler × Fayoumi and Broiler × Leghorn crosses respectively. These SNPs were on 10 chromosomes (GGA1, 2, 3, 4, 7, 8, 10, 12, 15 and 27). Interestingly, of the 39 SNPs that were significantly associated with AF, there were about twice as many homozygous genotypes associated with higher AF that traced back to the inbred lines alleles, although the broiler line had on average higher AF. These SNPs are considered to be associated with QTL with cryptic alleles. This study reveals cryptic alleles as an important factor in heterosis for fatness observed in two chicken F₂ populations, and suggests epistasis as the common underlying mechanism for heterosis and cryptic allele expression. The results of this study also demonstrate the power of high marker-density SNP association studies in discovering QTL that were not detected by previous microsatellite-based genotyping studies.     

The amino acid compositions of proteins are correlated with their molecular sizes.

Natural peptides and small proteins in general have amino acid compositions that diverge much more from the average composition of all proteins than do those of proteins. The effect is large and consistent enough to provide a rough check on the measured molecular mass of a protein and to indicate whether it is likely to have a significantly repetitive structure. For example, the alpha-chain of tropomyosin, a highly repetitive protein, has no amino acid composition that would be characteristic of a much smaller protein. The observation provides support for the suggestion [Taylor, Britton & van Heyningen (1983) Biochem. J. 209, 897-899] that tetanus toxin resembles a trimer of the light chain produced by proteolysis.     

Genome-wide mapping of DNA synthesis in Saccharomyces cerevisiae reveals that mechanisms preventing reinitiation of DNA replication are not redundant

To maintain genomic stability, reinitiation of eukaryotic DNA replication within a single cell cycle is blocked by multiple mechanisms that inactivate or remove replication proteins after G1 phase. Consistent with the prevailing notion that these mechanisms are redundant, we previously showed that simultaneous deregulation of three replication proteins, ORC, Cdc6, and Mcm2-7, was necessary to cause detectable bulk re-replication in G2/M phase in Saccharomyces cerevisiae. In this study, we used microarray comparative genomic hybridization (CGH) to provide a more comprehensive and detailed analysis of re-replication. This genome-wide analysis suggests that reinitiation in G2/M phase primarily occurs at a subset of both active and latent origins, but is independent of chromosomal determinants that specify the use and timing of these origins in S phase. We demonstrate that re-replication can be induced within S phase, but differs in amount and location from re-replication in G2/M phase, illustrating the dynamic nature of DNA replication controls. Finally, we show that very limited re-replication can be detected by microarray CGH when only two replication proteins are deregulated, suggesting that the mechanisms blocking re-replication are not redundant. Therefore we propose that eukaryotic re-replication at levels below current detection limits may be more prevalent and a greater source of genomic instability than previously appreciated.     

Signature proteins that are distinctive characteristics of Actinobacteria and their subgroups

The Actinobacteria constitute one of the main phyla of Bacteria. Presently, no morphological and very few molecular characteristics are known which can distinguish species of this highly diverse group. In this work, we have analyzed the genomes of four actinobacteria (viz. Mycobacterium leprae TN, Leifsonia xyli subsp. xyli str. CTCB07, Bifidobacterium longum NCC2705 and Thermobifida fusca YX) to search for proteins that are unique to Actinobacteria. Our analyses have identified 233 actinobacteria-specific proteins, homologues of which are generally not present in any other bacteria. These proteins can be grouped as follows: (i) 29 proteins uniquely present in most sequenced actinobacterial genomes; (ii) 6 proteins present in almost all actinobacteria except Bifidobacterium longum and another 37 proteins absent in B. longum and few other species; (iii) 11 proteins which are mainly present in Corynebacterium, Mycobacterium and Nocardia (CMN) subgroup as well as Streptomyces, T. fusca and Frankia sp., but they are not found in Bifidobacterium and Micrococcineae; (iv) 8 proteins that are specific for T. fusca and Streptomyces species, plus 2 proteins also present in the Frankia species; (v) 13 proteins that are specific for the Corynebacterineae or the CMN group; (vi) 14 proteins only found in Mycobacterium and Nocardia; (vii) 24 proteins unique to different Mycobacterium species; (viii) 8 proteins specific to the Micrococcineae; (ix) 85 proteins which are distributed sporadically in actinobacterial species. Additionally, many examples of lateral gene transfer from Actinobacteria to Magnetospirillum magnetotacticum have also been identified. The identified proteins provide novel molecular means for defining and circumscribing the Actinobacteria phylum and a number of subgroups within it. The distribution of these proteins also provides useful information regarding interrelationships among the actinobacterial subgroups. Most of these proteins are of unknown function and studies aimed at understanding their cellular functions should reveal common biochemical and physiological characteristics unique to either all actinobacteria or particular subgroups of them. The identified proteins also provide potential targets for development of drugs that are specific for actinobacteria.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Genome-wide analysis reveals that Smad3 and JMJD3 HDM co-activate the neural developmental program

Neural development requires crosstalk between signaling pathways and chromatin. In this study, we demonstrate that neurogenesis is promoted by an interplay between the TGFβ pathway and the H3K27me3 histone demethylase (HDM) JMJD3. Genome-wide analysis showed that JMJD3 is targeted to gene promoters by Smad3 in neural stem cells (NSCs) and is essential to activate TGFβ-responsive genes. In vivo experiments in chick spinal cord revealed that the generation of neurons promoted by Smad3 is dependent on JMJD3 HDM activity. Overall, these findings indicate that JMJD3 function is required for the TGFβ developmental program to proceed.     

The analysis of N-glycans of cell membrane proteins from human hematopoietic cell lines reveals distinctions in their pattern

Abstract Human cell lines are often different in their features and present variations in the glycosylation patterns of cell membrane proteins. Protein glycosylation is the most common posttranslational modification and plays a particular role in functionality and bioactivity. The key approach of this study is the comparative analysis of five hematopoietic cell lines for their N-glycosylation pattern. The N-glycans of membrane proteins were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF/TOF-MS analyses. Furthermore, the expression of a set of glycosyltransferases was determined via RT-PCR. The B-lymphoma BJA-B and promyelocytic HL-60 cell lines distinguish in levels and linkages of glycan-bound sialic acids. Furthermore, subclones of BJA-B and HL-60 cells, which completely lack UDP-N-acetylglucosamine 2-ēpimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis, contained almost no sialylated N-glycans. Compared to wild-type cells, the GNE-deficient cells pres\xadented a similar cell surface N-glycosylation pattern in terms of antennarity and fucosylation. The Jurkat T-cell line revealed only partially sialylated N-glycans. Additionally, the different hematopoietic cell lines vary in their level of bisecting GlcNAcylation and antennary fucosylation with the quantities of bisecting N-acetylglucosamine (GlcNAc) and core fucose coinciding with the expression of GnT-III and FucT-VIII. Of note is the occurrence of N-acetyllactosamine (LacNAc) extensions on tetraantennary structures in GNE-deficient cell lines.     

Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243

Burkholderia pseudomallei is an important human pathogen whose infection biology is still poorly understood. The bacterium is endemic to tropical regions, including South East Asia and Northern Australia, where it causes melioidosis, a serious disease associated with both high mortality and antibiotic resistance. B. pseudomallei is a Gram-negative facultative intracellular pathogen that is able to replicate in macrophages. However despite the critical nature of its interaction with macrophages, few anti-macrophage factors have been characterized to date. Here we perform a genome-wide gain of function screen of B. pseudomallei strain K96243 to identify loci encoding factors with anti-macrophage activity. We identify a total of 113 such loci scattered across both chromosomes, with positive gene clusters encoding transporters and secretion systems, enzymes/toxins, secondary metabolite, biofilm, adhesion and signal response related factors. Further phenotypic analysis of four of these regions shows that the encoded factors cause striking cellular phenotypes relevant to infection biology, including apoptosis, formation of actin 'tails' and multi-nucleation within treated macrophages. The detailed analysis of the remaining host of loci will facilitate genetic dissection of the interaction of this important pathogen with host macrophages and thus further elucidate this critical part of its infection cycle.     

Two-dimensional electrophoretic analysis reveals that lipid rafts are intact at physiological temperature

Different proteins are found in lipid rafts depending on the isolation method. For example, insulin receptor was predominantly found in lipid raft fractions prepared from HepG2 cells with Brij 35, but were not present in lipid rafts isolated with Triton X-100. In order to assess the effect of detergent type and temperature on raft isolation, raft proteins from HepG2 cells were analyzed by two-dimensional (2-D) electrophoresis. More raft protein spots appeared when rafts were isolated by Brij 35 than by Triton X-100. In addition, more raft proteins were found when isolated at 37 degrees C than at 4 degrees C, indicating that lipid rafts are much more stable at physiological temperature (37 degrees C) in the presence of detergents. Indeed, lipid-modified proteins, such as Src and Lyn, were found in raft fractions even when detergent-resistant rafts were isolated at room or physiological temperature. The 2-D gel profile of raft proteins isolated with detergent-free (high-pH/carbonate) method was considerably similar to that of detergent-resistant raft proteins but contained a greater number of distinct protein spots. Whereas many detergent-resistant raft proteins disappeared upon cellular exposure to methyl-beta-cyclodextrin, high pH/carbonate-resistant raft proteins did not, suggesting that many of proteins isolated by high pH/carbonate could be contaminants. Considering these data, we conclude that liquid-ordered state of detergent-resistant lipid rafts is not destroyed at physiological temperature.     

Statistical analysis of vertebrate sequences reveals that long genes are scarce in GC-rich isochores

We compared the exon/intron organization of vertebrate genes belonging to different isochore classes, as predicted by their GC content at third codon position. Two main features have emerged from the analysis of sequences published in GenBank: (1) genes coding for long proteins (i.e., 500 aa) are almost two times more frequent in GC-poor than in GC-rich isochores; (2) intervening sequences (=sum of introns) are on average three times longer in GC-poor than in GC-rich isochores. These patterns are observed among human, mouse, rat, cow, and even chicken genes and are therefore likely to be common to all warm-blooded vertebrates. Analysis of Xenopus sequences suggests that the same patterns exist in cold-blooded vertebrates. It could be argued that such results do not reflect the reality because sequence databases are not representative of entire genomes. However, analysis of biases in GenBank revealed that the observed discrepancies between GC-rich and GC-poor isochores are not artifactual, and are probably largely underestimated. We investigated the distribution of microsatellites and interspersed repeats in introns of human and mouse genes from different isochores. This analysis confirmed previous studies showing that Ll repeats are almost absent from GC-rich isochores. Microsatellites and SINES (Alu, B1, B2) are found at roughly equal frequencies in introns from all isochore classes. Globally, the presence of repeated sequences does not account for the increased intron length in GC-poor isochores. The relationships between gene structure and global genome organization and evolution are discussed.     

Mutant alleles that are altered in quantitative,organ-specific behavior

Three new mutant alleles of maize alcohol dehydrogenase-1 (Adh 1) were recovered following allyl alcohol selection of pollen. Each is altered in quantitative, organ-specific, regulatory properties. All mutant sites act in cis to the structural gene component. One mutant arose spontaneously, one followed indirectly from irradiation with high Z accelerated particles, and one was induced by an autonomous mutator system. Each mutant is assessed in three organs by utilizing ADH allozyme ratios that were quantified at the level of ADH enzyme activity and either [³H]-Leu incorporation into newly synthesized ADH 1 subunits or direct protein determinations. One mutation simultaneously raises Adh 1 expression in one organ and lowers it in another, another affects expression in one organ only, and another is extremely underexpressed in all organs but is unstable. This unstable allele has generated derivative mutant alleles that have less or zero ADH expression. We do not yet know whether or not coding sequences are involved in these mutants. We conclude that information for organ specificity and quantitative behavior resides near or within Adh 1 coding sequences.