Most of the murine leukemia virus sequences in the DNA of NIH/swiss mice consist of two closely related proviruses, each repeated several times

The structure of the endogenous murine leukemia virus (MuLV) sequences of NIH/Swiss mice was analyzed by restriction endonuclease digestion, gel electrophoresis, and hybridization to an MuLV nucleic acid probe. Digestion of mouse DNA with certain restriction endonucleases revealed two classes of fragments. A large number of fragments (about 30) were present at a relatively low concentration, indicating that each derived from a sequence present once in the mouse genome. A smaller number of fragments (one to five) were present at a much higher concentration and must have resulted from sequences present multiple times in the mouse genome. These results indicated that the endogenous MuLV sequences represent a family of dispersed repetitive sequences. Hybridization of these same digested mouse DNAs to nucleic acid probes representing different portions of the MuLV genome allowed construction of a map of the sites where restriction endonucleases cleave the endogenous MuLV sequences. Several independent recombinant DNA clones of endogenous MuLV sequences have been isolated from C3H mice (Roblin et al., J. Virol. 43:113-126, 1982). Analysis of these sequences shows that they have the structure of MuLV proviruses. The sites at which restriction endonucleases cleave within these proviruses appeared to be similar or identical to the sites at which these nucleases cleaved within the MuLV sequences of NIH/Swiss mice. This identity was confirmed by parallel electrophoresis. We conclude that the apparently complex pattern of endogenous MuLV sequences of NIH/Swiss mice consists largely of only two kinds of provirus, each repeated multiple times at dispersed sites in the mouse genome.     

Average genome size estimation improves comparative metagenomics and sheds light on the functional ecology of the human microbiome

Average genome size is an important, yet often overlooked, property of microbial communities. We developed MicrobeCensus to rapidly and accurately estimate average genome size from shotgun metagenomic data and applied our tool to 1,352 human microbiome samples. We found that average genome size differs significantly within and between body sites and tracks with major functional and taxonomic differences. In the gut, average genome size is positively correlated with the abundance of Bacteroides and genes related to carbohydrate metabolism. Importantly, we found that average genome size variation can bias comparative analyses, and that normalization improves detection of differentially abundant genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0611-7) contains supplementary material, which is available to authorized users.     

Mining the bovine genome with the "Bovine SNP Retriever"

Online resources for the bovine genome analysis are provided at the most important Web sites. Nonetheless, retrieval of single-nucleotide polymorphism (SNP)-related information is not always easy when searches must focus on complementary features. In this work, we present the Bovine SNP Retriever: a user-friendly tool for bovine SNP retrieval that also facilities the retrieval of SNP-related information within user-selected quantitative traits loci regions and reverse electronic polymerase chain reaction analysis on the bovine genome. The Bovine SNP Retriever is available at http://www.itb.cnr.it/ptp/bovine_snp_retriever/.     

A genome-wide analysis of FRT-like sequences in the human genome

Efficient and precise genome manipulations can be achieved by the Flp/FRT system of site-specific DNA recombination. Applications of this system are limited, however, to cases when target sites for Flp recombinase, FRT sites, are pre-introduced into a genome locale of interest. To expand use of the Flp/FRT system in genome engineering, variants of Flp recombinase can be evolved to recognize pre-existing genomic sequences that resemble FRT and thus can serve as recombination sites. To understand the distribution and sequence properties of genomic FRT-like sites, we performed a genome-wide analysis of FRT-like sites in the human genome using the experimentally-derived parameters. Out of 642,151 identified FRT-like sequences, 581,157 sequences were unique and 12,452 sequences had at least one exact duplicate. Duplicated FRT-like sequences are located mostly within LINE1, but also within LTRs of endogenous retroviruses, Alu repeats and other repetitive DNA sequences. The unique FRT-like sequences were classified based on the number of matches to FRT within the first four proximal bases pairs of the Flp binding elements of FRT and the nature of mismatched base pairs in the same region. The data obtained will be useful for the emerging field of genome engineering.     

Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome.

A system of versatile insertion plasmids was constructed that permits efficient delivery of the target sites of an ultra-rare-cutting endonuclease and the recombinase FLP into preselected sites of the bacterial genome. With the help of this system, the pathogenicity island LEE of the Escherichia coli O157:H7 genome was excised and isolated in vitro, deleted in vivo, rescued as a plasmid, and transferred into another strain.     

CpG Distribution and Methylation Pattern in Porcine Parvovirus

Based on GC content and the observed/expected CpG ratio (oCpGr), we found three major groups among the members of subfamily Parvovirinae: Group I parvoviruses with low GC content and low oCpGr values, Group II with low GC content and high oCpGr values and Group III with high GC content and high oCpGr values. Porcine parvovirus belongs to Group I and it features an ascendant CpG distribution by position in its coding regions similarly to the majority of the parvoviruses. The entire PPV genome remains hypomethylated during the viral lifecycle independently from the tissue of origin. In vitro CpG methylation of the genome has a modest inhibitory effect on PPV replication. The in vitro hypermethylation disappears from the replicating PPV genome suggesting that beside the maintenance DNMT1 the de novo DNMT3a and DNMT3b DNA methyltransferases can’t methylate replicating PPV DNA effectively either, despite that the PPV infection does not seem to influence the expression, translation or localization of the DNA methylases. SNP analysis revealed high mutability of the CpG sites in the PPV genome, while introduction of 29 extra CpG sites into the genome has no significant biological effects on PPV replication in vitro. These experiments raise the possibility that beyond natural selection mutational pressure may also significantly contribute to the low level of the CpG sites in the PPV genome.     

Mutational spectrum in the recent human genome inferred by single nucleotide polymorphisms

So far, there is no genome-wide estimation of the mutational spectrum in humans. In this study, we systematically examined the directionality of the point mutations and maintenance of GC content in the human genome using approximately 1.8 million high-quality human single nucleotide polymorphisms and their ancestral sequences in chimpanzees. The frequency of C-->T (G-->A) changes was the highest among all mutation types and the frequency of each type of transition was approximately fourfold that of each type of transversion. In intergenic regions, when the GC content increased, the frequency of changes from G or C increased. In exons, the frequency of G:C-->A:T was the highest among the genomic categories and contributed mainly by the frequent mutations at the CpG sites. In contrast, mutations at the CpG sites, or CpG-->TpG/CpA mutations, occurred less frequently in the CpG islands relative to intergenic regions with similar GC content. Our results suggest that the GC content is overall not in equilibrium in the human genome, with a trend toward shifting the human genome to be AT rich and shifting the GC content of a region to approach the genome average. Our results, which differ from previous estimates based on limited loci or on the rodent lineage, provide the first representative and reliable mutational spectrum in the recent human genome and categorized genomic regions.     

Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase

Currently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage and Flp from yeast. Both enzymes catalyze recombination between two 34-base pair recognition sites, lox and FRT, respectively, resulting in excision, inversion, or translocation of DNA sequences depending upon the location and the orientation of the recognition sites. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange. The problem with both recombinase systems is that when they insert a circular DNA into the genome (trans event), two cis-positioned recognition sites are created, which are immediate substrates for excision. To stabilize the trans event, functional mutant recognition sites had to be identified. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage phiC31 has been shown to function in Schizosaccharomyces pombe and mammalian cells. This enzyme recombines between two heterotypic sites: attB and attP. The product sites of the recombination event (attL and attR) are not substrates for the integrase. Therefore, the phiC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.     

Defining endogenous barcoding sites for CRISPR/Cas9-based cell lineage tracing in zebrafish

There is a growing interest in developing experimental methods for tracking the developmental cell lineages of a complex organism.The recently developed CRISPR/Cas9-based barcoding method is,although highly promising,difficult to scale up because it relies on exogenous barcoding sequences that are engineered into the genome.In this study,we characterized 78 high-quality endogenous sites in the zebrafish genome that can be used as CRISPR/Cas9-based barcoding sites.The 78 sites are all highly expressed in most of the cell types according to single-cell RNA sequencing(scRNA-seq)data.Hence,the barcoding information of the 78 endogenous sites is recovered by the available scRNA-seq platforms,enabling simultaneous characterization of cell type and cell lineage information.