In vitro assay for inhibitors of the intron self-splicing reacti

Details In vitro assay for inhibitors of the intron self-splicing reacti In vitro assay for inhibitors of the intron self-splicing reacti

1995-06-19

1998-12-15

Myers, Carla J.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 9121, 435 915, 435 9151, 435 9153, 435184, 435195, 536 2433, 935 8, 935 78, 935 17, 935 18, C12Q 168, C12P 1934, C07H 2104

Patent

active

058494847

ABSTRACT:
The present invention pertains to an in vitro method for assaying for an inhibitor of the catalytic Group I self-splicing intron reaction in the nuclear rRNA genes of Pheumocystis carinii which comprises the steps of (a) providing a DNA template containing the intron (I) from the 26S rRNA gene in Pneumocystis carinii and a portion of the 5' and 3' flanking exons (E1 and E2, respectively) between nucleotides 1963 and 2267 of 26S rRNA (660 nucleotides of amplified rRNA gene including the group I intron); (b) preparing an RNA precursor by transcription of the DNA template in the presence of labeled nucleoside triphosphates to produce a labeled RNA precursor (E1-I-E2); (c) purifying the RNA precursor; (d) incubating the RNA precursor and the inhibitor in the presence of guanosine triphosphate and magnesium ions; and (e) determining the degree of inhibition by the inhibitor on the intron splicing reaction in the RNA precursor by measuring the amount of labeled splicing intermediates and splicing products.

REFERENCES:
Lin et al Gene (1992, Oct.) 119: 163-173.
Liu et al Nucleic Acids Research (1992, Jul.) 20: 3763-3772.

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