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DNA microarrays allow the massively parallel, semiquantitative analysis of gene expression at the whole-genome level. Fabricated by robots that deposit near-microscopic spots of DNA onto solid surfaces, a single microarray can carry tens of thousands of unique DNA fragments. By exploiting DNAs ability to form highly specific base pairs, such microarrays allow samples to be checked for the presence and relative abundance of each DNA fragment represented on the array surface.
In an elegant method first reported by Patrick Brown (Stanford University), two samples can be characterized simultaneously with respect to their relative amounts of mRNA using a two-color fluorescence microarray assay. This approach allows researchers to determine how specific environmental conditions affect gene expression at the level of transcription.
Observing Radiation Effects
Microarrays containing over 12,000 human cDNAs fabricated in the state-of-the-art facility at Los Alamos National Laboratory (LANL) are providing a fruitful platform for understanding the cells response to low doses of direct and indirect ionizing radiation. Using primary human cell culture and acute radiation doses, Robert Carys laboratory at LANL is applying bioinformatics approaches to discover new regulatory pathways responsible for transcriptional response. In collaboration with Bruce Lehnert (LANL), investigators are exploring the response to reactive oxygen species generated as a by-product of ionizing radiation exposure. This work will point the way to identifying key gene products, biochemical pathways, and regulatory networks responsible for DNA damage recognition and repair, cell-cycle checkpoints, and other metabolic responses that determine individual susceptibility or resistance to ionizing radiation.
Understanding Anthrax Infection
LANL scientists also are using DNA microarrays to better understand microbial virulence, as in Bacillus anthracis, the cause of anthrax and thus a key threat as a biological weapon. B. anthracis is poorly understood with respect to the molecular mechanisms of pathogenesis, which is known to require the presence of the two large virulence plasmids pX01 and pX02. Using LANL-fabricated microarrays and an attenuated B.anthracis strain incapable of causing the disease, investigators have characterized the expression of 143 predicted open reading frames (ORFs) on the pX01 plasmid. Although this plasmid contains many predicted ORFs, only four previously had responded at the transcriptional level to changes in temperature and carbonate concentrationtwo manipulable environmental parameters that can mimic conditions encountered by the microbe during infection. Interestingly, these four ORFs play central roles in pathogenesis, which suggests that identifying additional plasmid ORFs with similar expression profiles will provide important new clues to the molecular mechanisms of B. anthracis pathogenesis.
Focusing on the expression pattern of 143 pX01 ORFs, a series of studies has been conducted to examine transcription-level changes in response to temperature and carbonate. Experiments conducted at LANL by Cary and James Pannucci, a postdoctoral fellow working with Cheryl Kuske, have revealed marked changes in RNA levels for a large number of ORFs. Strikingly, these experiments have revealed that 35 pX01 ORFs, in addition to the 4 previously characterized plasmid genes, respond to increases in temperature and carbonate concentration by increasing gene transcription. These ORFS provide an exciting starting point for follow-up studies designed to characterize their role in B. anthracis virulence. Data from these studies undoubtedly will play a fundamental role in revealing new key factors in B. anthracis pathogenesis.
Robert B. Cary, LANL
The electronic form of the newsletter may be cited in the following style:
Human Genome Program, U.S. Department of Energy, Human Genome News (v12n1-2).
The Human Genome Project (HGP) was an international 13-year effort, 1990 to 2003. Primary goals were to discover the complete set of human genes and make them accessible for further biological study, and determine the complete sequence of DNA bases in the human genome. See Timeline for more HGP history.