Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Retroviral restriction factor APOBEC3G delays the initiation of DNA synthesis by HIV-1 reverse transcriptase.

It is well established that the cytosine deaminase APOBEC3G can restrict HIV-1 virions in the absence of the virion infectivity factor (Vif) by inducing genome mutagenesis through deamination of cytosine to uracil in single-stranded HIV-1 (-)DNA. However, whether APOBEC3G is able to restrict HIV-1 using a deamination-independent mode remains an open question. In this report we use in vitro primer extension assays on primer/templates that model (-)DNA synthesis by reverse transcriptase from the primer binding site (PBS) and within the protease gene of HIV-1. We find that APOBEC3G is able to decrease the initiation of DNA synthesis by reverse transcriptase approximately 2-fold under conditions where reverse transcriptase is in excess to APOBEC3G, as found in HIV-1 virions. However, the delay in the initiation of DNA synthesis on RNA templates up to 120 nt did not decrease the total amount of primer extended after extended incubation unless the concentration of reverse transcriptase was equal to or less than that of APOBEC3G. By determining apparent Kd values of reverse transcriptase and APOBEC3G for the primer/templates and of reverse transcriptase binding to APOBEC3G we conclude that APOBEC3G is able to decrease the efficiency of reverse transcriptase-mediated DNA synthesis by binding to the RNA template, rather than by physically interacting with reverse transcriptase. All together the data support a model in which this deamination-independent mode of APOBEC3G would play a minor role in restricting HIV-1. We propose that the deamination-independent inhibition of reverse transcriptase we observed can be a mechanism used by APOBEC3G to slow down proviral DNA formation and increase the time in which single-stranded (-)DNA is available for deamination by APOBEC3G, rather than a direct mechanism used by APOBEC3G for HIV-1 restriction

In situ mass spectrometry imaging reveals heterogeneous glycogen stores in human normal and cancerous tissues

Abstract Glycogen dysregulation is a hallmark of aging, and aberrant glycogen drives metabolic reprogramming and pathogenesis in multiple diseases. However, glycogen heterogeneity in healthy and diseased tissues remains largely unknown. Herein, we describe a method to define spatial glycogen architecture in mouse and human tissues using matrix‐assisted laser desorption/ionization mass spectrometry imaging. This assay provides robust and sensitive spatial glycogen quantification and architecture characterization in the brain, liver, kidney, testis, lung, bladder, and even the bone. Armed with this tool, we interrogated glycogen spatial distribution and architecture in different types of human cancers. We demonstrate that glycogen stores and architecture are heterogeneous among diseases. Additionally, we observe unique hyperphosphorylated glycogen accumulation in Ewing sarcoma, a pediatric bone cancer. Using preclinical models, we correct glycogen hyperphosphorylation in Ewing sarcoma through genetic and pharmacological interventions that ablate in vivo tumor growth, demonstrating the clinical therapeutic potential of targeting glycogen in Ewing sarcoma

Trypanosome RNA Editing: Simple Guide RNA Features Enhance U Deletion 100-Fold

Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro U deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3′ OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing