ALBERT

Description: Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N 6 -adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N 6 -adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5'-CGY m6 A G-3'), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5'-AC m6 A CC-3') and ModD1 (exemplified by M.Nme579I) (5'-CC m6 A GC-3'). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5'-CGY m6 A G-3'. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5'-GCGC m6 A GG-3' sites, to 100% at 5'-ACGT m6 A GG-3' sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.

Description: We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes.

Description: We identify a new subgroup of Type I Restriction-Modification enzymes that modify cytosine in one DNA strand and adenine in the opposite strand for host protection. Recognition specificity has been determined for ten systems using SMRT sequencing and each recognizes a novel DNA sequence motif. Previously characterized Type I systems use two identical copies of a single methyltransferase (MTase) subunit, with one bound at each half site of the specificity (S) subunit to form the MTase. The new m4C-producing Type I systems we describe have two separate yet highly similar MTase subunits that form a heterodimeric M1M2S MTase. The MTase subunits from these systems group into two families, one of which has NPPF in the highly conserved catalytic motif IV and modifies adenine to m6A, and one having an NPPY catalytic motif IV and modifying cytosine to m4C. The high degree of similarity among their cytosine-recognizing components (MTase and S) suggest they have recently evolved, most likely from the far more common m6A Type I systems. Type I enzymes that modify cytosine exclusively were formed by replacing the adenine target recognition domain (TRD) with a cytosine-recognizing TRD. These are the first examples of m4C modification in Type I RM systems.

Description: Aim The ability to quantitatively measure the continuum of macroscale patterns of species invasion is a first step toward deeper understanding of their causal factors. We took advantage of two centuries worth of herbarium data, to evaluate a set of metrics to measure macroscale patterns, allowing cross-species comparisons of invasive expansion across large geographic areas. Methods We used herbarium specimens to reconstruct county-level invasion histories for two non-native plants ( Alliaria petiolata and Lonicera japonica ), with distinct spatiotemporal distribution patterns over the past two centuries. Using county centroids from species’ initial occurrences, we quantified point pattern metrics from multiple disciplines (e.g. urban crime analysis, landscape ecology etc.) that are historically used at smaller spatial scales, to evaluate their ability to detect macroscale spatial diffusion and amount of directional expansion. Metrics were further assessed for their ease of use, data requirements, independence from other metrics and intuitiveness of interpretation. Important Findings We identified four suitable metrics for distinguishing differences in spatial patterns: (i) standard distance, (ii) number of patches, (iii) Euclidean nearest neighbor summary class statistic coefficient of variation and (iv) mean center that when applied to county-level presence data allowed us to determine the directions by which distributions expanded and if distributions increased via outward expansion, infilling and/or jump dispersal events. These metrics when compared during the same invasion phase are capable of quantifying macroscale variability among species in their distributional and dispersal patterns. Being able to quantify differences among species in these patterns is important in understanding the drivers of species dispersal patterns. These metrics therefore represent a simple yet thorough toolset for achieving this goal.

Description: Base J (β-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae . We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication.