did not affect the exponential growth rate but did confer a strong selective disadvantage on the mutant when it was competed against wild-type. The negative selection did not appear to be acting at either the exponential or stationary phase. Transformation with a plasmid vector conferring carbenicillin resistance and growth in carbenicillin markedly increased the selective disadvantage, as did growth at 42 8C, and both together were approximately additive such that three cycles of competitive growth sufficed to reduce the mutant strain to ;0.2% of its original value. The most striking finding was that all growth effects could be reversed by transformation with a plasmid carrying a truB gene coding for a D48C mutation in TruB. Direct analysis showed that this mutant did not make C55 under the conditions of the competition experiment. Therefore, the growth defect due to the lack of TruB must be due to the lack of some other function of the protein, possibly an RNA chaperone activity, but not to the absence of C55.

RluD is the pseudouridine synthase responsible for the formation of C1911, C1915, and C1917 in Escherichia coli 23S rRNA. Previous work from our laboratory demonstrated that disruption of the rluD gene and/or loss of the pseudouridine residues for which it is responsible resulted in a severe growth phenotype. In the current work we have examined further the effect of the loss of the RluD protein and its product pseudouridine residues in a deletion strain lacking the rluD gene. This strain exhibits defects in ribosome assembly, biogenesis, and function. Specifically, there is a deficit of 70S ribosomes, an increase in 50S and 30S subunits, and the appearance of new 62S and 39S particles. Analysis of the 39S particles indicates that they are immature precursors of the 50S subunits, whereas the 62S particles are derived from the breakdown of unstable 70S ribosomes. In addition, purified mutant 70S ribosomes were found to be somewhat less efficient than wild type in protein synthesis. The defect in ribosome assembly and resulting growth phenotype of the mutant could be restored by expression of wild-type RluD and synthesis of C1911, C1915, and C1917 residues, but not by catalytically inactive mutant RluD proteins, incapable of pseudouridine formation. The data suggest that the loss of the pseudouridine residues can account for all aspects of the mutant phenotype; however, a possible second function of the RluD synthase is also discussed.

psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes Um2552 in E. coli, makes the 50S subunit less stable at 1 mM Mg++ (Bügl et al. 2000) and inhibits subunit joining (Caldas et al. 2000), but, in this case, it is not yet known whether the effects are due to the lack of 2'-O-methylation or to the absence of the enzyme itself. Is there any role for the psi residues themselves? First, as noted above, the 3 psi made by RluD which cluster in the end-loop of helix 69 are highly conserved, with one being universal (Fig. 2B). In the 70S-tRNA structure (Yusupov et al. 2001), the loop of this helix containing the psi supports the anticodon arm of A-site tRNA near its juncture with the amino acid arm. The middle of helix 69 does the same thing for P-site tRNA. Unfortunately, the resolution is not yet sufficient to provide a more precise alignment of the psi residues with the other structural elements of the tRNA-ribosome complex so that one cannot yet determine what role, if any, is played by the N-1 H that distinguishes psi from U. Second, and more generally, some psi residues in the LSU appear to be near the sit...

This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines. Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.

An example is presented of an IgG1, anti-Lu6, that reacted by indirect antiglobulin test and was capable of destroying antigen-positive red cells in vivo. Two methods for the measurement of red cell survival, 51Cr labeling and flow cytometry, gave the same result: 20 percent of the test dose of Lu:6 red cells was destroyed in the first hour after injection and 80 percent in the first 24 hours. The clinical relevance of the antibody was correctly predicted by an in vitro monocyte monolayer assay. The finding that this example of anti-Lu6 was clinically significant should not be taken to mean that all antibodies directed against high-incidence Lutheran and Lutheran system-related antigens will behave similarly. When such antibodies are encountered, in vivo and/or in vitro studies to assess their clinical significance are necessary before rare blood is used for transfusion.