Effect of 5′UTR introns on gene expression in Arabidopsis thaliana

Background The majority of introns in gene transcripts are found within the coding sequences (CDSs). A small but significant fraction of introns are also found to reside within the untranslated regions (5′UTRs and 3′UTRs) of expressed sequences. Alignment of the whole genome and expressed sequence tags (ESTs) of the model plant Arabidopsis thaliana has identified introns residing in both coding and non-coding regions of the genome. Results A bioinformatic analysis revealed some interesting observations: (1) the density of introns in 5′UTRs is similar to that in CDSs but much higher than that in 3′UTRs; (2) the 5′UTR introns are preferentially located close to the initiating ATG codon; (3) introns in the 5′UTRs are, on average, longer than introns in the CDSs and 3′UTRs; and (4) 5′UTR introns have a different nucleotide composition to that of CDs and 3′UTR introns. Furthermore, we show that the 5′UTR intron of the A. thaliana EFIα-A3 gene affects the gene expression and the size of the 5′UTR intron influences the level of gene expression. Conclusion Introns within the 5′UTR show specific features that distinguish them from introns that reside within the coding sequence and the 3′UTR. In the EFIα-A3 gene, the presence of a long intron in the 5′UTR is sufficient to enhance gene expression in plants in a size dependent manner.

Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma

The imprinted gene, neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report, we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localization studies of normal pituitaries showed that each of the hormone secreting cells (GH, PRL, ACTH, FSH and TSH) expressed NNAT. However, 33 out of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by quantitative real-time RT-PCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where approximately 50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT, relative to normal pituitaries, the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT-20 cells significantly reduced CFE. Collectively, these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.

Global dissemination of a multidrug resistant Escherichia coli clone

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000–2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL–resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.

Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Variation in the OC locus of Acinetobacter baumannii genomes predicts extensive structural diversity in the Lipooligosaccharide

Lipooligosaccharide (LOS) is a complex surface structure that is linked to many pathogenic properties of Acinetobacter baumannii. In A. baumannii, the genes responsible for the synthesis of the outer core (OC) component of the LOS are located between ilvE and aspS. The content of the OC locus is usually variable within a species, and examination of 6 complete and 227 draft A. baumannii genome sequences available in GenBank non-redundant and Whole Genome Shotgun databases revealed nine distinct new types, OCL4-OCL12, in addition to the three known ones. The twelve gene clusters fell into two distinct groups, designated Group A and Group B, based on similarities in the genes present. OCL6 (Group B) was unique in that it included genes for the synthesis of L-Rhamnosep. Genetic exchange of the different configurations between strains has occurred as some OC forms were found in several different sequence types (STs). OCL1 (Group A) was the most widely distributed being present in 18 STs, and OCL6 was found in 16 STs. Variation within clones was also observed, with more than one OC locus type found in the two globally disseminated clones, GC1 and GC2, that include the majority of multiply antibiotic resistant isolates. OCL1 was the most abundant gene cluster in both GC1 and GC2 genomes but GC1 isolates also carried OCL2, OCL3 or OCL5, and OCL3 was also present in GC2. As replacement of the OC locus in the major global clones indicates the presence of sub-lineages, a PCR typing scheme was developed to rapidly distinguish Group A and Group B types, and to distinguish the specific forms found in GC1 and GC2 isolates.

Four generations of sequencing: Is it ready for the clinic yet?

Next-generation sequencing techniques have revolutionized over the last decade providing researchers with low cost, high-throughput alternatives compared to the traditional Sanger sequencing methods. These sequencing techniques have rapidly evolved from first-generation to fourth-generation with very broad applications such as unravelling the complexity of the genome, in terms of genetic variations, and having a high impact on the biological field. In this review, we discuss the transition of sequencing from the second-generation to the third- and fourth-generations, and describe some of their novel biological applications. With the advancement in technology, the earlier challenges of minimal size of the instrument, flexibility of throughput, ease of data analysis and short run times are being addressed. However, the need for prospective analysis and effectiveness to test whether the knowledge of any given new variants identified has an effect on clinical outcome may need improvement.

Identity-by-Descent Mapping to Detect Rare Variants Conferring Susceptibility to Multiple Sclerosis

Genome-wide association studies (GWAS) have identified around 60 common variants associated with multiple sclerosis (MS), but these loci only explain a fraction of the heritability of MS. Some missing heritability may be caused by rare variants that have been suggested to play an important role in the aetiology of complex diseases such as MS. However current genetic and statistical methods for detecting rare variants are expensive and time consuming. 'Population-based linkage analysis' (PBLA) or so called identity-by-descent (IBD) mapping is a novel way to detect rare variants in extant GWAS datasets. We employed BEAGLE fastIBD to search for rare MS variants utilising IBD mapping in a large GWAS dataset of 3,543 cases and 5,898 controls. We identified a genome-wide significant linkage signal on chromosome 19 (LOD = 4.65; p = 1.9×10-6). Network analysis of cases and controls sharing haplotypes on chromosome 19 further strengthened the association as there are more large networks of cases sharing haplotypes than controls. This linkage region includes a cluster of zinc finger genes of unknown function. Analysis of genome wide transcriptome data suggests that genes in this zinc finger cluster may be involved in very early developmental regulation of the CNS. Our study also indicates that BEAGLE fastIBD allowed identification of rare variants in large unrelated population with moderate computational intensity. Even with the development of whole-genome sequencing, IBD mapping still may be a promising way to narrow down the region of interest for sequencing priority. © 2013 Lin et al.

Expression profiling in spondyloarthropathy synovial biopsies highlights changes in expression of inflammatory genes in conjunction with tissue remodelling genes

Background: In the spondyloarthropathies, the underlying molecular and cellular pathways driving disease are poorly understood. By undertaking a study in knee synovial biopsies from spondyloarthropathy (SpA) and ankylosing spondylitis (AS) patients we aimed to elucidate dysregulated genes and pathways. Methods RNA was extracted from six SpA, two AS, three osteoarthritis (OA) and four normal control knee synovial biopsies. Whole genome expression profiling was undertaken using the Illumina DASL system, which assays 24000 cDNA probes. Differentially expressed candidate genes were then validated using quantitative PCR and immunohistochemistry. Results: Four hundred and sixteen differentially expressed genes were identified that clearly delineated between AS/SpA and control groups. Pathway analysis showed altered gene-expression in oxidoreductase activity, B-cell associated, matrix catabolic, and metabolic pathways. Altered «myogene» profiling was also identified. The inflammatory mediator, MMP3, was strongly upregulated (5-fold) in AS/SpA samples and the Wnt pathway inhibitors DKK3 (2.7-fold) and Kremen1 (1.5-fold) were downregulated. Conclusions: Altered expression profiling in SpA and AS samples demonstrates that disease pathogenesis is associated with both systemic inflammation as well as local tissue alterations that may underlie tissue damaging modelling and remodelling outcomes. This supports the hypothesis that initial systemic inflammation in spondyloarthropathies transfers to and persists in the local joint environment, and might subsequently mediate changes in genes directly involved in the destructive tissue remodelling.

Identification of major loci controlling clinical manifestations of ankylosing spondylitis

Objective. To identify genomic regions linked with determinants of age at symptom onset, disease activity, and functional impairment in ankylosing spondylitis (AS). Methods. A whole genome linkage scan was performed in 188 affected sibling pair families with 454 affected individuals. Traits assessed were age at symptom onset, disease activity assessed by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and functional impairment assessed by the Bath Ankylosing Spondylitis Functional Index (BASFI). Parametric and nonparametric quantitative linkage analysis was performed using parameters defined in a previous segregation study. Results. Heritabilities of the traits studied in this data set were as follows: BASDAI 0.49 (P = 0.0001, 95% confidence interval [95% CI] 0.23-0.75), BASFI 0.76 (P = 10-7, 95% CI 0.49-1.0), and age at symptom onset 0.33 (P = 0.005, 95% CI 0.04-0.62). No linkage was observed between the major histocompatibility complex (MHC) and any of the traits studied (logarithm of odds [LOD] score <1.0). "Significant" linkage (LOD score 4.0) was observed between a region on chromosome 18p and the BASDAI. Age at symptom onset showed "suggestive" linkage to chromosome 11p (LOD score 3.3). Maximum linkage with the BASFI was seen at chromosome 2q (LOD score 2.9). Conclusion. In contrast to the genetic determinants of susceptibility to AS, clinical manifestations of the disease measured by the BASDAI, BASFI, and age at symptom onset are largely determined by a small number of genes not encoded within the MHC.

Dissimilarities in alignment-free methods for phylogenetic analysis based on genomes

Whole genome sequences are generally accepted as excellent tools for studying evolutionary relationships. Due to the problems caused by the uncertainty in alignment, existing tools for phylogenetic analysis based on multiple alignments could not be directly applied to the whole-genome comparison and phylogenomic studies. There has been a growing interest in alignment-free methods for phylogenetic analysis using complete genome data. The “distances” used in these alignment-free methods are not proper distance metrics in the strict mathematical sense. In this study, we first review them in a more general frame — dissimilarity. Then we propose some new dissimilarities for phylogenetic analysis. Last three genome datasets are employed to evaluate these dissimilarities from a biological point of view.

The revolution in human monogenic disease mapping

The successful completion of the Human Genome Project (HGP) was an unprecedented scientific advance that has become an invaluable resource in the search for genes that cause monogenic and common (polygenic) diseases. Prior to the HGP, linkage analysis had successfully mapped many disease genes for monogenic disorders; however, the limitations of this approach were particularly evident for identifying causative genes in rare genetic disorders affecting lifespan and/or reproductive fitness, such as skeletal dysplasias. In this review, we illustrate the challenges of mapping disease genes in such conditions through the ultra-rare disorder fibrodysplasia ossificans progressiva (FOP) and we discuss the advances that are being made through current massively parallel (“next generation”) sequencing (MPS) technologies.

Convergent evolution of marine mammals is associated with distinct substitutions in common genes

Phenotypic convergence is thought to be driven by parallel substitutions coupled with natural selection at the sequence level. Multiple independent evolutionary transitions of mammals to an aquatic environment offer an opportunity to test this thesis. Here, whole genome alignment of coding sequences identified widespread parallel amino acid substitutions in marine mammals; however, the majority of these changes were not unique to these animals. Conversely, we report that candidate aquatic adaptation genes, identified by signatures of likelihood convergence and/or elevated ratio of nonsynonymous to synonymous nucleotide substitution rate, are characterized by very few parallel substitutions and exhibit distinct sequence changes in each group. Moreover, no significant positive correlation was found between likelihood convergence and positive selection in all three marine lineages. These results suggest that convergence in protein coding genes associated with aquatic lifestyle is mainly characterized by independent substitutions and relaxed negative selection.

Brain-specific epigenetic markers of schizophrenia

Epigenetics plays a crucial role in schizophrenia susceptibility. In a previous study, we identified over 4500 differentially methylated sites in prefrontal cortex (PFC) samples from schizophrenia patients. We believe this was the first genome-wide methylation study performed on human brain tissue using the Illumina Infinium HumanMethylation450 Bead Chip. To understand the biological significance of these results, we sought to identify a smaller number of differentially methylated regions (DMRs) of more functional relevance compared with individual differentially methylated sites. Since our schizophrenia whole genome methylation study was performed, another study analysing two separate data sets of post-mortem tissue in the PFC from schizophrenia patients has been published. We analysed all three data sets using the bumphunter function found in the Bioconductor package minfi to identify regions that are consistently differentially methylated across distinct cohorts. We identified seven regions that are consistently differentially methylated in schizophrenia, despite considerable heterogeneity in the methylation profiles of patients with schizophrenia. The regions were near CERS3, DPPA5, PRDM9, DDX43, REC8, LY6G5C and a region on chromosome 10. Of particular interest is PRDM9 which encodes a histone methyltransferase that is essential for meiotic recombination and is known to tag genes for epigenetic transcriptional activation. These seven DMRs are likely to be key epigenetic factors in the aetiology of schizophrenia and normal brain neurodevelopment.

Sequence variants in three loci influence monocyte counts and erythrocyte volume

Blood cells participate in vital physiological processes, and their numbers are tightly regulated so that homeostasis is maintained. Disruption of key regulatory mechanisms underlies many blood-related Mendelian diseases but also contributes to more common disorders, including atherosclerosis. We searched for quantitative trait loci (QTL) for hematology traits through a whole-genome association study, because these could provide new insights into both hemopoeitic and disease mechanisms. We tested 1.8 million variants for association with 13 hematology traits measured in 6015 individuals from the Australian and Dutch populations. These traits included hemoglobin composition, platelet counts, and red blood cell and white blood cell indices. We identified three regions of strong association that, to our knowledge, have not been previously reported in the literature. The first was located in an intergenic region of chromosome 9q31 near LPAR1, explaining 1.5% of the variation in monocyte counts (best SNP rs7023923, p=8.9x10(-14)). The second locus was located on chromosome 6p21 and associated with mean cell erythrocyte volume (rs12661667, p=1.2x10(-9), 0.7% variance explained) in a region that spanned five genes, including CCND3, a member of the D-cyclin gene family that is involved in hematopoietic stem cell expansion. The third region was also associated with erythrocyte volume and was located in an intergenic region on chromosome 6q24 (rs592423, p=5.3x10(-9), 0.6% variance explained). All three loci replicated in an independent panel of 1543 individuals (p values=0.001, 9.9x10(-5), and 7x10(-5), respectively). The identification of these QTL provides new opportunities for furthering our understanding of the mechanisms regulating hemopoietic cell fate.

Phylogenetic analysis of human Chlamydia pneumoniae strains reveals a distinct Australian indigenous clade that predates European exploration of the continent

Background The obligate intracellular bacterium Chlamydia pneumoniae is a common respiratory pathogen, which has been found in a range of hosts including humans, marsupials and amphibians. Whole genome comparisons of human C. pneumoniae have previously highlighted a highly conserved nucleotide sequence, with minor but key polymorphisms and additional coding capacity when human and animal strains are compared. Results In this study, we sequenced three Australian human C. pneumoniae strains, two of which were isolated from patients in remote indigenous communities, and compared them to all available C. pneumoniae genomes. Our study demonstrated a phylogenetically distinct human C. pneumoniae clade containing the two indigenous Australian strains, with estimates that the most recent common ancestor of these strains predates the arrival of European settlers to Australia. We describe several polymorphisms characteristic to these strains, some of which are similar in sequence to animal C. pneumoniae strains, as well as evidence to suggest that several recombination events have shaped these distinct strains. Conclusions Our study reveals a greater sequence diversity amongst both human and animal C. pneumoniae strains, and suggests that a wider range of strains may be circulating in the human population than current sampling indicates.