IDENTIFICATION OF DIFFERENTIALLY EXPRESSED MESSENGER-RNA IN PROKARYOTIC ORGANISMS BY CUSTOMIZED AMPLIFICATION LIBRARIES (DECAL) - THE EFFECT OF ISONIAZID ON GENE-EXPRESSION IN MYCOBACTERIUM-TUBERCULOSIS

Understanding the effects of the external environment on bacterial gen e expression can provide valuable insights into an array of cellular m echanisms including pathogenesis, drug resistance, and, in the case of Mycobacterium tuberculosis, latency, Because of the absence of poly(A )(+) mRNA in prokaryotic organisms, studies of differential gene expre ssion currently must be performed either with large amounts of total R NA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an app roach to study differences in bacterial mRNA expression that enables a mplification by the PCR of a complex mixture of cDNA sequences in a re producible manner that obviates the confounding effects of selected hi ghly expressed sequences, e,g,, ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of am plifiable genomic sequences to convert total cellular RNA into an ampl ified probe for gene expression screens. DECAL can detect 4-fold diffe rences in the mRNA levels of rare sequences and can be performed on as little as 10 ng of total RNA. DECAL was used to investigate the in vi tro effect of the antibiotic isoniazid on M, tuberculosis, and three p reviously uncharacterized isoniazid-induced genes, iniA, iniB, and ini C, were identified, The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestiv e of an acyl carrier protein. The iniA gene is also induced by the ant ibiotic ethambutol, an agent that inhibits cell wall biosynthesis by a mechanism that is distinct from isoniazid, The DECAL method offers a powerful new tool for the study of differential gene expression.