Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.

Myxococcus xanthus, a gram-negative soil bacterium, responds to amino acid starvation by entering a process of multicellular development which culminates in the assembly of spore-filled fruiting bodies. Previous studies utilizing developmental inhibitors (such as methionine, lysine, or threonine) have revealed important clues about the mechanisms involved in fruiting body formation. We used Biolog phenotype microarrays to screen 384 chemicals for complete inhibition of fruiting body development in M. xanthus. Here, we report the identification of a novel inhibitor of fruiting body formation and sporulation, ␤-D-allose. ␤-D-Allose, a rare sugar, is a member of the aldohexose family and a C3 epimer of glucose. Our studies show that ␤-D-allose does not affect cell growth, viability, agglutination, or motility. However, ␤-galactosidase reporters demonstrate that genes activated between 4 and 14 h of development show significantly lower expression levels in the presence of ␤-D-allose. Furthermore, inhibition of fruiting body formation occurs only when ␤-D-allose is added to submerged cultures before 12 h of development. In competition studies, high concentrations of galactose and xylose antagonize the nonfruiting response to ␤-D-allose, while glucose is capable of partial antagonism. Finally, a magellan-4 transposon mutagenesis screen identified glcK, a putative glucokinase gene, required for ␤-D-allose-mediated inhibition of fruiting body formation. Subsequent glucokinase activity assays of the glcK mutant further supported the role of this protein in glucose phosphorylation.Although Myxococcus xanthus exists as individual vegetative rods, the myxobacteria are fundamentally social organisms capable of intercellular communication, coordinated group movements, and the formation of multicellular structures. Upon starvation, a developmental program is initiated wherein bacterial cells aggregate into dome-shaped, macroscopic structures called fruiting bodies. The signaling and genetic systems orchestrating this program have received much attention in M. xanthus research (21,22,47). In particular, investigation of exogenous compounds that inhibit fruiting body formation have provided useful starting points for identifying the signaling pathways and molecular components involved in this highly regulated process. Previous studies investigating inhibition of fruiting body formation by various amino acids have shown that methionine-mediated inhibition is associated with a dramatic reduction in the production of S-adenosylmethionine (SAM) (6, 42). SAM, a methyl donor for the M. xanthus frz chemosensory system, affects fruiting through the regulation of motility (5, 45). Lysine, threonine, leucine, and isoleucine were also found to inhibit fruiting body formation through a reduction of intracellular levels of SAM. A separate study found that glycine can block purine-induced fruiting body formation, suggesting that purine-containing compounds, such as cyclic AMP and ADP, induce fruiting through a single mechanism (38).To identif...