N6-methyladenosine (m6A) has been shown to be the most common base modification in eukaryotic messenger RNA (mRNA) other than the 7-methylguanosine cap. Recent m6A-RNA immunoprecipitation (m6A-RIP) with polyclonal antibodies combined with RNA high-throughput sequencing (RNA-seq) studies have identified the location of m6A sites in a transcriptome-wide manner in a variety of tissues and have started to analyze the function of m6A in mRNA (1-8). In humans m6A is most commonly associated with a sequence motif in the 3’ UTR of mRNAs near stop codons and m6A modification is dependent upon a complex consisting of the methyltranferases METTL3+METTL14 and accessory proteins such as WTAP and KIAA1429.
To further advance our understanding of m6A in RNA, it is important to continue improving the tools needed for m6A research. Here we present the generation of a new m6A-specific rabbit monoclonal antibody and its use in m6A-RIP-seq experiments.
The role of prokaryotic DNA methyltransferases within restriction-modification systems has been well established, while the functional role of the many orphan DNA methyltransferases is still far from clear. Two examples of Dam- and Dcm- orphan DNA methyltransferases have been extensively studied and functional roles in mismatch DNA repair, DNA replication and phase variation of protein expression have been established.
We took advantage of the recently developed platform for single-molecule real time sequencing by Pacific Biosciences to investigate DNA methyltransferase specificity. The analysis of total DNA from two pathogenic strains of B. cenocepacia J2315 and E.coli O104:H4 genomic DNA has revealed the presence of two unusual methyltransferases not previously characterized. Both are plasmid-encoded by ORFs in pBCA072 for B. cenocepacia J2315 and pESBL for E.coli O104:H4. They both result in single-stranded, almost non-specific m6A modification, within the motif SAB (where S = C or G and B = C, G or T). This methylation is partial and only detected on plasmid DNA. We have called these enzymes M.BceJIII and M.EcoGIX respectively.
A set of genetic and biochemical experiments suggested that the activity of these enzymes is associated with plasmid replication and depended on the origin of replication. While ColEI and p15 origins support plasmid modification, the pSC101 origin does not. Moreover, we demonstrated that these enzymes work as a complex with DNA polymerase I during plasmid replication and may modify the lagging strand. It is possible they control plasmid and phage replication by discriminating DNA polymerase I-dependent and non-dependent plasmids origins. We suggest that the base flipping inherent to DNA modification may allow the methylase to perform a DNA helicase function and thereby help to control the rate of DNA polymerization to prevent excessive recombination.
The taxonomy of Beggiatoa genus is still a work in progress. Despite many morphotypes of the Beggiatoa genus having been described in the literature, only one species, B. alba has been validated until now. In 2016, we described a second species, B. leptomitoformis. Two strains of B. leptomitoformis D401 and D402 have been isolated from different regions of Russia. They differ in their morphology and physiology especially their ability to growth lithotrophically in the presence of thiosulfate. While B. leptomitoformis D402 is able to accumulate elemental sulfur, strainD401 cannot. We performed genomic sequencing of these two strains using the PacBio SMRT platform and assembled the reads into two complete circular genomes: B. leptomitiformis D-401 with 4,266,286 bp and D-402 with 4,265,296 bp. Both genome sequences have been deposited in GenBank with accession numbers CP018889 and CP012373 respectively (1). Surprisingly these two genomes showed almost 99% identity. The preliminary analysis of several thiosulfate oxidation (,soxA, soxF and soxW) and autotrophic assimilation of CO2 (cbbQ, rbcL) metabolic operones in both stains did not revel any noticeable differences.
The advantage of the PacBio sequencing platform is its ability to detect the epigenetic state of the sequenced DNA, which allows for the identification of modified nucleotides and the corresponding motifs in which they occur. Thirteen DNA methyltransferase recognition motifs were found. They include one m4C and nine m6A modifications that were detected by direct SMRT sequencing and an additional three m5C motifs were detected in Tet2 treated DNA. The motifs were then matched with methyltransferase genes in the genome, and the results have been deposited in REBASE (2).
Accurate quantitation of a next-generation sequencing library is essential to maximizing data output and quality from each instrument run. qPCR is widely accepted as the most effective method for library quantitation by both users and manufacturers, as qPCR methods measure only sequenceable library fragments with a high level of accuracy and consistency. The NEBNext Library Quant Kit for Illumina presents a simple, robust method for quantitation of Illumina libraries. Here we demonstrate the effectiveness of the Kit for a broad range of library types and sizes as well as advantages offered by qPCR quantitation for obtaining optimal cluster density and user-to-user consistency. The NEBNext Quant Kit offers an efficient and cost-effective qPCR library quantitation workflow for users looking to optimize both sequencing yield and throughput.
The ability to produce high levels of recombinant protein has become one of the cornerstones of biological sciences. Purified proteins allow us to obtain information about their specificities and even structures. To date, many proteins have been expressed and purified from engineered host cells, such as E. coli, due to the relative simplicity of the process. Unfortunately, many other proteins of interest are not readily expressed or expressed as insoluble aggregates in bacterial hosts. Many advances have been made over the past several decades to understand and improve folding and solubility of recombinant proteins within their new hosts, however, there are still many enzymes that do not fold well.
Each protein is unique and we believe that creating customized expression strains for each difficult protein is a strategy that is currently under-utilized, in part because of the time and effort required for extensive genome modification in E. coli. Here, we present the development of a novel, rapid method for E. coli genome engineering, allowing us to make markerless genome modifications in 48 hours. As an example of such modification, we restored wild-type lon allele in a B strain and demonstrate its beneficial effect on production of a membrane protein.
Ligation of two adjacent DNA oligonucleotides splinted by RNA has historically been difficult to achieve. We have discovered that SplintR® ligase (Chlorella virus DNA ligase) is much more efficient in this ligation than T4 DNA Ligase, which was traditionally used for this application. When these ligases were compared using the same RNA:DNA substrates the SplintR® enzyme achieved complete ligation with 10X less ligase and was 15X faster than T4 DNA Ligase .
The characterization of glycoprotein structure is becoming increasingly sophisticated, as regulatory agencies require multiple attributes to be measured during development, production, and formulation of biological drugs. Precise determination of N- and O-glycosylation, site occupancy, disulfide shuffling, misassembly, deamidation, oxidation, etc, require robust methods for sample preparation, to facilitate mass spectrometry analysis. Enzymes for glycan removal, along with specific proteases, are critical to these studies. Improved methods where glycosidases are combined, and/or coupled with labeling reactions or protease digestion, maximize reproducibility by eliminating handling errors. These methods, in turn, permit a more stringent definition of an original, biosimilar, or biobetter, facilitating formulation and process development innovations. We present in this poster new glycan removal protocols, including fast deglycosylation (using Rapid PNGase F) and deglycosylation of intact plant-derived glycoproteins (using PNGase Ar). These reactions were coupled with a simplified and versatile glycan labeling reaction by reductive amination, suitable for glycans lacking a glycosylamine end group. Also, glycosidase combinations were tested for complete N- and O-glycan removal, to facilitate proteomic analysis for glycoproteins that are heavily glycosylated. Finally, an enzyme mix containing PNGase F and Trypsin was used to prepare an IgG sample for peptide mapping. This abbreviated workflow maintained sensitivity and reproducibility
During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications and generation of doublestranded DNA breaks.To address the components required for the process, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed.
Uses hemimethylated DNA as substrate.
SwaI, a Type IIP restriction enzyme from Staphylococcus warneri cleaves the symmetric sequence ATTT|AAAT, producing fragments with blunt ends (‘|’ = cleavage site). We solved the crystal structure of SwaI alone, of SwaI bound to uncleaved DNA in the presence of Ca2+ ions, and of SwaI bound to cleaved DNA in the presence of Mg2+ ions. We describe these structures, and compare them to that of PacI, which cleaves the related 8-bp sequence, TTAAT|TAA.
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