Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3 ′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.
Contents: ,,Madura cattle," the variety found on the Indonesian island of Madura, is most often referred t o as a cross between Bos javanicus and Bos indicus, based largely on phenotypic appearance. The karyotypic patterns of Madura cattle resemble those o f Bos taurus, with the exception of the Y chromosome, which is of Bos indicus type. Based on what is known of Bos javanicus, it is concluded that Madura cattle could be the result of a cross between a Bos taurus or Bos javanicus cow and a Bos indicus bull. Inhalt: Eine cytogenetische Untersuchung iiber das Madura-Rind Das Madura-Rind, eine Varietat der indonesischen Znsel Madura, wird wegen seines Aussehens oft als Kreuzung zwischen Bos javanicus (Banteng) und Bos indicus (Zebu) angesehen. Das karyotypische Bild des Madura-Rindes gleicht jenem von Bos taurus mit Ausnahme des Y-Chromosoms, welches dem Bos indicus-Typ entspricht. Nachdem, was iiber Bos javanicus bekannt ist, mu& man folgern, dafl das Madura-Rind ein Kreuzungsprodukt zwischen einer Bos taurus-oder Bos javanicus-Kuh und einem Bos indi-cusBullen sein konnte.
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