21 Genome-Wide Approaches to Studying Prokaryotic Biology

Transcriptional profiling using DNA microarrays. Shown is an outline of a two-color labeling scheme, with the objective to determine the transcriptome of a microorganism grown under two different conditions. A spotted microarray is first generated by PCR-amplifying all open reading frames in the organism's annotated genome, and spotting them onto derivatized microscope slides using a robotic arrayer. The bacteria are grown under two different conditions, and total RNA is isolated from each culture. These are then converted to labeled target by first-strand cDNA synthesis, with simultaneous incorporation of Cy3 or Cy5 nucleotides. The two labeled targets are combined and cohybridized to the probes on the microarray. Following hybridization and washing, the amount of target bound to each probe is determined by scanning the array and recording the fluorescence of Cy3 and Cy5 after excitation at their respective wavelength, which gives maximal emission. The relative intensities reflect the abundance of the mRNAs present in each sample, and the relative amount of bound target is usually displayed by pseudocolor representation. Red represents excess binding of Cy5 over Cy3, while green indicates that this probe bound more Cy3 than Cy5. Yellow indicates that equal amounts of Cy3 and Cy5 bound to each probe, and that the corresponding gene is not differentially regulated.

Construction of a DNA microarray by photolithographic synthesis of oligonucleotide probes. Probes corresponding to small (∼25 nucleotide) portions of genes can be synthesized by sequential light-induced deprotection of nucleotides and subsequent addition of nucleotides. Each deprotection step necessitates the use of a mask, which exposes only those probes that need to be deprotected and serve as acceptors of the next nucleotide. This method allows high-density synthesis of up to a million probes per array, with each gene represented by 10 to 20 probes.

Methods of labeling targets for DNA microarray. (A) Incorporation of Cy3 or Cy5 nucleotides during DNA synthesis. (B) A two-step labeling procedure, whereby amine-modified nucleotides are incorporated during cDNA synthesis. The cDNA is then labeled by covalent linkage of N-hydroxysuccinimide Cy3 or Cy5.

Amplification of target signal on the microarray. Biotin-labeled cDNA target is generated by incorporating biotin-modified nucleotides during cDNA synthesis or by attaching a biotinylated nucleotide to the 5′ end of the cDNA using terminal transferase. The bound cDNA is detected by sequential application of streptavidin, biotinylated antistreptavidin antibody, and fluorescently labeled streptavidin.

Genome-wide location analysis. Genome-wide location analysis is a DNA microarray application designed to identify transcription factor binding sites. First, protein-DNA complexes within cells are cross-linked with formaldehyde. The cross-linked complexes are isolated by immunoprecipitation with specific antibody, and the DNA is released by reversing the cross-links. The DNA is then amplified by PCR to generate targets for microarray analysis. A corresponding sample is generated from total DNA that has not been subjected to immunoprecipitation. The signal ratio derived from the two samples identifies sequences that are directly bound by specific factors, thus demonstrating direct regulation of those genes by those factors.

Transposon site hybridization analysis (TraSH). Replicate pools of mutants are grown under two different conditions, and differentially labeled target is produced from the chromosomal sequences flanking each transposon in each pool. The two labeled targets are cohybridized to a mircroarray, and the signal ratio at each spot quantitates the representation of that sequence in the two pools. TraSH thus provides information about which genes are required for survival under a given condition.

Applications of isotope-coded affinity tag (ICAT) technology. Protein samples, such as lysates from bacteria grown under two different conditions, are chemically derivatized with the isotopically light or heavy version of the ICAT reagent. The labeled samples are combined and proteolyzed to yield small peptide fragments. The derivatized fragments are isolated by affinity chromatography, using a tag that is part of the ICAT reagent. The isolated peptides are separated, identified, and quantified following liquid chromatography (LC) and tandem mass spectrometry (MS).

Two protein array formats. (A) Protein-detecting array. A set of capture reagents, usually antibodies specific for each protein analyzed, is immobilized on a solid surface. A protein mixture is applied, and bound protein is detected using a second reagent, such as another specific antibody. (B) Protein function array. A set of expressed proteins is immobilized in an array format on a solid support. These can be then used in a variety of assays to study enzymatic activity or interaction with substrates, including other proteins or nucleic acids.