The Purification of Small RNAs by High-Performance Liquid Chromatography

Dudock, Bernard S.

doi:10.1016/b978-0-12-372483-0.50024-7

Cited by 1 publication

(1 citation statement)

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“…Ten to 20 mg of total tRNA can be isolated from 200 g of human placenta by using the method of Roe (32), which uses standard phenol-chloroform extractions to isolate total RNA, followed by DEAE-cellulose chromatography to isolate total tRNA. The tRNA was aminoacylated with [3H]lysine, and tRNALYS fractions were isolated by using standard chromatographic methods for tRNA purification (sequentially, DEAE-Sephadex A-50, reverse-phase chromatography [RPC-5], and Porex C4 chromatography [8,29]). A final purification step used two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) as described below.…”

Section: Methodsmentioning

confidence: 99%

Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1

Jiang

Mak

Ladha

et al. 1993

J Virol

189

109

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We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain HIB (HIV-111,B) produced in COS-7 cells transfected with HlV-1 proviral DNA and mutant, noninfectious HIV-1L, particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNALYS, tRNAI"Y (the putative primer for HIV-1 reverse transcriptase) and tRNA". Identification was accomplished by comparing the electrophoretic mobilities and RNase T, digests with those of tRNA3LY' and tRNAj', purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNALYS incorporation into HIV-1. However, only the wild-type virus contains tRNALY" tightly associated with the viral RNA genome. The identification of the * Corresponding author.

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Section: Methodsmentioning

confidence: 99%