Identification of differentially expressed in mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in Mycobacterium tuberculosis

D. Alland, I. Kramnik, T. R. Weisbrod, L. Otsubo, R. Cerny, L. P. Miller, Jr Jacobs W.R., B. R. Bloom

Research output: Contribution to journalArticle

87 Citations (Scopus)

Abstract

Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms 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 expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens. DECAL can detect 4-fold differences 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 vitro effect of the antibiotic isoniazid on M. tuberculosis, and three previously uncharacterized isoniazid-induced genes, iniA, iniB, and iniC, were identified. The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein. The iniA gene is also induced by the antibiotic 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.

Original languageEnglish (US)
Pages (from-to)13227-13232
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume95
Issue number22
DOIs
StatePublished - Oct 27 1998

Fingerprint

Isoniazid
Mycobacterium tuberculosis
Libraries
Gene Expression
Messenger RNA
RNA
Cell Wall
Acyl Carrier Protein
Genes
Anti-Bacterial Agents
Bacterial Genes
Ethambutol
Ribosomal RNA
Genomic Library
Complex Mixtures
Drug Resistance
Complementary DNA
Polymerase Chain Reaction
Proteins

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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title = "Identification of differentially expressed in mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in Mycobacterium tuberculosis",
abstract = "Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms 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 expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens. DECAL can detect 4-fold differences 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 vitro effect of the antibiotic isoniazid on M. tuberculosis, and three previously uncharacterized isoniazid-induced genes, iniA, iniB, and iniC, were identified. The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein. The iniA gene is also induced by the antibiotic 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.",
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T2 - The effect of isoniazid on gene expression in Mycobacterium tuberculosis

AU - Alland, D.

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AU - Weisbrod, T. R.

AU - Otsubo, L.

AU - Cerny, R.

AU - Miller, L. P.

AU - Jacobs W.R., Jr

AU - Bloom, B. R.

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