Labeling of eukaryotic messenger RNA 5' terminus with phosphorus -32: use of tobacco acid pyrophosphatase for removal of cap structures

Journal Article · Thu Jan 01 00:00:00 EST 1981 · Gene Amplif. Anal.; (United States)

OSTI ID:5904878

Lockard, R E; Rieser, L; Vournakis, J N

In recent years, there has been a growing appreciation of the potential applications of 5'-/sup 32/P-end-labeled mRNA, not only for screening recombinant clones and mapping gene structure, but also for revealing possible nucleotide sequence and structural signals within mRNA molecules themselves, which may be important for eukaryotic mRNA processing and turnover and for controlling differential rates of translational initiation. Three major problems, however, have retarded progress in this area, lack of methods for efficient and reproducible removal of m7G5ppp5'-cap structures, which maintain the integrity of an RNA molecule; inability to generate a sufficient amount of labeled mRNA, owing to the limited availability of most pure mRNA species; and the frequent problem of RNA degradation during in vitro end-labeling owing to RNAse contamination. The procedures presented here permit one to decap and label minute quantities of mRNA, effectively. Tobacco acid pyrophosphatase is relatively efficient in removing cap structures from even nanogram quantities of available mRNA, and enough radioactivity can be easily generated from minute amounts ofintact mRNA with very high-specific-activity (gamma-/sup 32/P)ATP and the inhibition of ribonuclease contamination with diethylpyrocarbonate. These procedures can be modified and applied to almost any other type of RNA molecule as well. In Section III of this volume, we explore in detail how effectively 5'-end-labeled mRNA can be used not only for nucleotide sequence analysis, but also for mapping mRNA secondary structure.

• Research Organization: George Washington Univ. Medical School, Washington, DC (USA)
• OSTI ID: 5904878
• Journal Information: Gene Amplif. Anal.; (United States), Vol. 2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
MESSENGER-RNA
LABELLING
PHOSPHATASES
BIOLOGICAL FUNCTIONS
ATP
GENETIC MAPPING
HYDROLYSIS
MOLECULAR STRUCTURE
PHOSPHORUS 32
RNA-ASE
TOBACCO
TRACER TECHNIQUES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ENZYMES
ESTERASES
FUNCTIONS
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LYSIS
MAPPING
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS ISOTOPES
RADIOISOTOPES
RNA
SOLVOLYSIS
550201* - Biochemistry- Tracer Techniques