A method is described for the large-scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol a-toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor-rich membrane fragments is composed of only one class of membrane fragments covered with 8-nm rosettes identified as acetylcholine receptor molecules.This preparation is used as a starting material for the detergent solubilization and the large-scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X-100 extract of receptor-rich membranes done in the presence of 2-mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy.Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecyl sulfate reveals three bands of apparent molecular weights 40 000

Conservation of microRNAs (miRNAs) among species suggests that they bear conserved biological functions. However, sequencing of new miRNAs has not always been accompanied by a search for orthologues in other species. I report herein the results of a systematic search for interspecies orthologues of miRNA precursors, leading to the identification of 35 human and 45 mouse new putative miRNA genes. MicroRNA tracks were written to visualize miRNAs in human and mouse genomes on the UCSC Genome Browser. Based on their localization, miRNA precursors can be excised either from introns or exons of mRNAs. When intronic miRNAs are antisense to the apparent host gene, they appear to originate from ill‐characterized antisense transcription units. Exonic miRNAs are, in general, nonprotein‐coding, poorly conserved genes in sense orientation. In three cases, the excision of an miRNA from a protein‐coding mRNA might lead to the degradation of the rest of the transcript. Moreover, three new examples of miRNAs fully complementary to an mRNA are reported. Among these, miR135a might control the stability and/or translation of an alternative form of the glycerate kinase mRNA by RNA interference. I also discuss the presence of human miRNAs in introns of paralogous genes and in miRNA clusters.

MicroRNAs are tiny RNA molecules that play important regulatory roles in a broad range of developmental, physiological or pathological processes. Despite recent progress in our understanding of miRNA processing and biological functions, little is known about the regulatory mechanisms that control their expression at the transcriptional level. C19MC is the largest human microRNA gene cluster discovered to date. This 100-kb long cluster consists of 46 tandemly repeated, primate-specific pre-miRNA genes that are flanked by Alu elements (Alus) and embedded within a ∼400- to 700-nt long repeated unit. It has been proposed that C19MC miRNA genes are transcribed by RNA polymerase III (Pol-III) initiating from A and B boxes embedded in upstream Alu repeats. Here, we show that C19MC miRNAs are intron-encoded and processed by the DGCR8-Drosha (Microprocessor) complex from a previously unidentified, non-protein-coding Pol-II (and not Pol-III) transcript which is mainly, if not exclusively, expressed in the placenta.