We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenosine, 5-methylcytosine, and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of the primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We utilize these kinetic signatures to identify adenosine methylation in genomic samples and show that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns within even highly repetitive genomic regions.

The integrase from the Streptomyces phage C31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the C31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.

Plants have unique features that evolved in response to their environments and ecosystems. A full account of the complex cellular networks that underlie plant-specific functions is still missing. We describe a proteome-wide binary protein-protein interaction map for the interactome network of the plant Arabidopsis thaliana containing ~6,200 highly reliable interactions between ~2,700 proteins. A global organization of plant biological processes emerges from community analyses of the resulting network, together with large numbers of novel hypothetical functional links between proteins and pathways. We observe a dynamic rewiring of interactions following gene duplication events, providing evidence for a model of evolution acting upon interactome networks. This and future plant interactome maps should facilitate systems approaches to better understand plant biology and improve crops.

We previously established that the phage C31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.For the past 25 years, it has been possible to construct precisely designed DNA molecules in the test tube thanks to the techniques of recombinant DNA. In contrast, the ability to make controlled and efficient alterations in the genomes of living higher cells has been limited. The use of site-specific recombinases such as Cre and FLP provided an important advance (17), but because of the reversibility of these enzyme reactions, their main utility has been for creating deletions. For the integration of new material into the genome, fortuitous integration of transfected DNA is most often used, and it produces integration at random locations at low frequency. Homologous recombination provides site specificity, but at very low efficiency (26).We began working with another site-specific recombinase, the phage C31 integrase, because it offered the potential for unidirectional integration that would therefore occur at higher net frequencies than the reversible integration directed by recombinases, such as Cre. Cre recombines two identical loxP sites, recreating two identical sites after recombination that can undergo a subsequent round of recombination. In contrast, the attB and attP recognition sites recognized by the C31 integrase are dissimilar in sequence (15). After reaction, the recombined att sites differ from attB and attP and are refractory to further synapsis by the integrase, thus locking in integration reactions (23,24). We demonstrated that this enzyme, derived from a Streptomyces phage (9), worked well in the human cell environment (7), consistent with its lack of cofactor requirements (23). This feature distinguishes it from better known phage integrases, such as that of phage , which does require cofactors (10). The integrase is in the family of recombinases that includes Cre and FLP and carries out a tyrosine-mediated strand exchange (4, 13). The C31 integrase is in the other major family of site-specific recombinases that includes many resolvases and invertases and uses a serine-catalyzed reaction mechanism (20). The two site-specific recombinase families are unrelated. The C31 integrase is a member of a recently discovered subclass of the serine recombinase family whose members are especia...

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