Processing of insect retrotransposons by self-cleaving ribozymes

We show that several classes of insect non-LTR retrotransposons harbor self-cleaving ribozymes of the HDV family at their 5′ termini. In Drosophila the R2 ribozymes exhibit highly differential in vivo expression and robust in vitro activity, modulated by an upstream sequence originating from the insertion site. Our data suggest a role for self-cleaving ribozymes in co-transcriptional processing of retrotransposons with implications for downstream events, including translation and retrotransposition

ICAR: endoscopic skull‐base surgery

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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

Kinetic Parameters of trans Scission by Extended HDV-like Ribozymes and the Prospect for the Discovery of Genomic trans-Cleaving RNAs.

Hepatitis delta virus (HDV)-like ribozymes are self-cleaving catalytic RNAs with a widespread distribution in nature and biological roles ranging from self-scission during rolling-circle replication in viroids to co-transcriptional processing of eukaryotic retrotransposons, among others. The ribozymes fold into a double pseudoknot with a common catalytic core motif and highly variable peripheral domains. Like other self-cleaving ribozymes, HDV-like ribozymes can be converted into trans-acting catalytic RNAs by bisecting the self-cleaving variants at non-essential loops. Here we explore the trans-cleaving activity of ribozymes derived from the largest examples of the ribozymes (drz-Agam-2 family), which contain an extended domain between the substrate strand and the rest of the RNA. When this peripheral domain is bisected at its distal end, the substrate strand is recognized through two helices, rather than just one 7 bp helix common among the HDV ribozymes, resulting in stronger binding and increased sequence specificity. Kinetic characterization of the extended trans-cleaving ribozyme revealed an efficient trans-cleaving system with a surprisingly high KM', supporting a model that includes a recently proposed activation barrier related to the assembly of the catalytically competent ribozyme. The ribozymes also exhibit a very long koff for the products (∼2 weeks), resulting in a trade-off between sequence specificity and turnover. Finally, structure-based searches for the catalytic cores of these ribozymes in the genome of the mosquito Anopheles gambiae, combined with sequence searches for their putative substrates, revealed two potential ribozyme-substrate pairs that may represent examples of natural trans-cleaving ribozymes

Preparation of splicing competent nuclear extracts.

Splicing components play an essential role in mediating accurate and efficient splicing. The complexity of the spliceosome and its regulatory networks increase the difficulty of studying the splicing reaction in detail. Nuclear extracts derived from HeLa cells provide all of the obligatory components to carry out intron removal in vitro. This chapter describes the large-scale preparation of nuclear extract from HeLa cells