Portal de Periódicos da CAPES

[14] Characterization of cap structures

1989; Academic Press; Linguagem: Inglês

10.1016/0076-6879(89)80100-4

1557-7988

Yasuhiro Furuichi, Aaron J. Shatkin,

Bacteriophages and microbial interactions

This chapter reviews the methods, which were developed for the preparation of capped mRNAs, and the isolation and characterization of different cap molecules. Several eukaryotic viruses, such as human reovirus, vaccinia virus, and insect cytoplasmic polyhedrosis virus (CPV), contain as components of the purified virus particle enzymes that catalyze the synthesis of capped mRNAs, including RNA polymerase, guanylyltransferase, and methyltransferase(s). The selective radiolabeling of specific sites in caps can be planned on the basis of the mechanism of synthesis. After incubation, virions are removed by centrifugation in an Eppendorf microcentrifuge, and the RNAs in the supernatant fraction are isolated by phenol extraction and gel filtration on a column of Sephadex G-100 or by standard affinity chromatography on oligo(dT)-cellulose. Caps obtained by enzymatic digestion of mRNAs elute from diethylaminoethyl (DEAE)-cellulose columns between 0.2 and 0.25 M NaCI; for cap isolation, fractions from this region of a linear salt gradient are combined.