Prospective analysis of adverse events in surgical treatment of degenerative spondylolisthesis.

BACKGROUND CONTEXT: Surgical adverse event (AE) monitoring is imprecise, of uncertain validity, and tends toward underreporting. Reports focus on specific procedures rather than outcomes in the context of presenting diagnosis. Specific intraoperative (intraop) or postoperative (postop) AEs that may be independently associated with degenerative spondylolisthesis (DS) have never been reported. PURPOSE: The primary purpose was to assess the AE profile of surgically treated patients with L4-L5 DS. The secondary goal was to identify potential risk factors that correlate with those AEs. STUDY DESIGN/SETTING: Prospective cohort and academic quaternary spine center. PATIENT SAMPLE: Ninety-two patients with L4-L5 DS were treated surgically, discharged from Vancouver General Hospital between January 1, 2009 and December 31, 2010. OUTCOME MEASURES: Incidence rates and odds ratios. METHODS: Prospective AE data were analyzed using univariate analyses, forward selection regression models, and Spearman correlation coefficients. Results were compared with outcomes reported in the Spine Patient Outcomes Research Trial. RESULTS: No AEs were seen in 57.6% of patients, one AE in 17.4%, and two or more AEs in 17.4%. Dural tears (6.5%) and intraop bone-implant interface failure requiring revision (3.3%) were the most common intraop AEs. Postoperatively, the most frequent AEs were urinary tract infection (10.9%), delirium (5.4%), neuropathic pain (4.4%), deep wound infection (3.3%), and superficial wound infection (3.3%). The odds of an intraop AE increased by 9% (95% confidence interval [CI] 1-18) per year of age at admission. Adjusted Charlson comorbidity index (CCI) did not correlate with number of AEs experienced. The odds of postop delirium correlated with CCI (odds ratio [OR] 3.39, 95% CI 1.12-10.24) and dural tear (OR 35.84, 95% CI 1.72-747.45). Length of stay was statistically significant and was influenced by two or more AEs, CCI, postop loss of correction, cerebrospinal fluid leak, deep wound infection, noninfected wound drainage, and gender. CONCLUSIONS: Risk of intraop AEs, but not postop AEs, increased with increasing age. Having multiple comorbidities does not predispose to more AEs. Infections predominate among the postop AEs. Patients at increased risk of delirium or of having an increased length of hospital stay may more easily be predicted. Studies specifically designed to prospectively assess AEs have the potential to more accurately identify postop AE rates.

Maximum rrn promoter activity in Escherichia coli at saturating concentrations of free RNA polymerase.

During fast growth, the rrn P1 promoters of Escherichia coli operate at their maximum strength, but below their maximum activity (V(max)), since they are not saturated with RNA polymerase. Since higher concentrations of free RNA polymerase are expected to be found in strains carrying rrn deletions, we have analyzed reported electron micrographs of rrn operons from rrn deletion strains growing at maximal rates (at 37 degrees C) in LB medium [1]. We conclude that, in a strain with four of the seven rrn operons inactivated by partial deletions, transcripts are initiated at rrn P1 promoters 1.6-fold more rapidly than in the wild-type strain and the entirety of the rrn operon is transcribed at a 1.5-fold higher average elongation rate due to shortened pauses in the 16S and 23S regions. Under this condition, traffic congestion occurs in front of a pause site in the 5' leader region of the rrn operon near the beginning of the 16S gene; the congestion extends all the way back to the promoter, impedes promoter clearance and limits the promoter activity to one initiation per 0.56 s. This corresponds to a promoter activity of 107 transcripts/min and is assumed to be close to the V(max) of rrn P1 promoters.

Varying rate of RNA chain elongation during rrn transcription in Escherichia coli.

The value of the rRNA chain elongation rate in bacteria is an important physiological parameter, as it affects not only the rRNA promoter activity but also the free-RNA polymerase concentration and thereby the transcription of all genes. On average, rRNA chains elongate at a rate of 80 to 90 nucleotides (nt) per s, and the transcription of an entire rrn operon takes about 60 s (at 37 degrees C). Here we have analyzed a reported distribution obtained from electron micrographs of RNA polymerase molecules along rrn operons in E. coli growing at 2.5 doublings per hour (S. Quan, N. Zhang, S. French, and C. L. Squires, J. Bacteriol. 187:1632-1638, 2005). The distribution exhibits two peaks of higher polymerase density centered within the 16S and 23S rRNA genes. An evaluation of this distribution indicates that RNA polymerase transcribes the 5' leader region at speeds up to or greater than 250 nt/s. Once past the leader, transcription slows down to about 65 nt/s within the 16S gene, speeds up in the spacer region between the 16S and 23S genes, slows again to about 65 nt/s in the 23S region, and finally speeds up to a rate greater than 400 nt/s near the end of the operon. We suggest that the slowing of transcript elongation in the 16S and 23S sections is the result of transcriptional pauses, possibly caused by temporary interactions of the RNA polymerase with secondary structures in the nascent rRNA.

A guided tour: small RNA function in Archaea.

In eukaryotes, the C/D box family of small nucleolar (sno)RNAs contain complementary guide regions that are used to direct 2'-O-ribose methylation to specific nucleotide positions within rRNA during the early stages of ribosome biogenesis. Direct cDNA cloning and computational genome searches have revealed homologues of C/D box snoRNAs (called sRNAs) in prokaryotic Archaea that grow at high temperature. The guide sequences within the sRNAs indicate that they are used to direct methylation to nucleotides in both rRNAs and tRNAs. The number of sRNA genes that are detectable within currently sequenced genomes correlates with the optimal growth temperature. We suggest that archaeal sRNAs may have two functions: to guide the deposition of methyl groups at the 2'-O position of ribose, which is an important determinant in RNA structural stability, and to serve as a molecular chaperones to help orchestrate the folding of rRNAs and tRNAs at high temperature.

Genome sequence of Halobacterium species NRC-1.

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

Homologs of small nucleolar RNAs in Archaea.

In eukaryotes, dozens of posttranscriptional modifications are directed to specific nucleotides in ribosomal RNAs (rRNAs) by small nucleolar RNAs (snoRNAs). We identified homologs of snoRNA genes in both branches of the Archaea. Eighteen small sno-like RNAs (sRNAs) were cloned from the archaeon Sulfolobus acidocaldarius by coimmunoprecipitation with archaeal fibrillarin and NOP56, the homologs of eukaryotic snoRNA-associated proteins. We trained a probabilistic model on these sRNAs to search for more sRNAs in archaeal genomic sequences. Over 200 additional sRNAs were identified in seven archaeal genomes representing both the Crenarchaeota and the Euryarchaeota. snoRNA-based rRNA processing was therefore probably present in the last common ancestor of Archaea and Eukarya, predating the evolution of a morphologically distinct nucleolus.

Transcriptional analysis of the glutamate dehydrogenase gene in the primitive eukaryote, Giardia lamblia. Identification of a primordial gene promoter.

We studied gene expression in the ancient eukaryote, Giardia lamblia, by taking advantage of assays developed recently in our laboratory, which allow new genetic analyses of this organism. We examined the transcription of a 2.2-kilobase segment of the Giardia genome that contains the glutamate dehydrogenase (GDH) gene and a portion of a second open reading frame encoding an uncharacterized gene. Nuclear run-on analyses showed that the genes are transcribed as two separate units spaced less than 200 base pairs apart, and transcription of the GDH gene initiates just 3-6 nucleotides upstream of its translation start codon. We characterized the GDH promoter by transfecting Giardia with DNA constructs that used the GDH upstream sequence to drive the expression of a luciferase reporter gene. By deletion and mutational analyses, we localized promoter function to three motifs within a 50-base pair region of the GDH upstream sequence. Using band shift assays and UV cross-linking, we demonstrated specific binding of a 68-kDa protein from Giardia nuclear extracts to short poly(T) tracts contained within two of the sequence motifs on single-stranded DNA from the promoter region. This report describes one of the first functional gene promoter and its cognate DNA-binding protein in this primitive eukaryote.

Substrate requirements for a novel archaeal endonuclease that cleaves within the 5' external transcribed spacer of Sulfolobus acidocaldarius precursor rRNA.

During ribosome biogenesis in the hyperthermophilic archaeon Sulfolobus acidocaldarius, at least three separate precursor endonucleolytic cleavages occur within the 144-nucleotide-long 5' external transcribed spacer (5' ETS) region of the rRNA operon primary transcript. The 5' ETS sequence contains three regions of very stable helical structure. One cleavage (5' to position -98) is in the single-stranded region between the 5' and the central helical domains; a second cleavage (5' to position -31) is in the single-stranded region between the central and the 3' helical domains; and a third cleavage is at the 5' ETS-16S junction (5' to position +1). The three sites share a common consensus sequence around the position of cleavage. We have used an in vitro pre-RNA processing assay to define some of the sequence and structural recognition elements necessary for the two precursor cleavages 5' to positions -98 and -31. Surprisingly, none of the three predominant helical domains are required for recognition or targeting of the cleavages, although their removal reduces the rate of cleavage site utilization. We show that the sequence AAG downward arrow (CA)UU encompassing each site contains at least some of the essential features for recognition and efficient targeting of the cleavages. Cleavage depends on the presence of a purine 5' and a uracil two nucleotides 3' to the scissile phosphodiester bond. Mutations to other bases at these critical positions are either not cleaved or cleaved very poorly. Finally, on the basis of intermediates that are produced during a processing reaction, we can conclude that the cleavages at positions 98 and 31 are not ordered in vitro.

RNA polymerase of Aquifex pyrophilus: implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria.

A 16,226-bp fragment from the genome of Aquifex pyrophilus was sequenced, containing the genes for ribosomal proteins L1, L10, and L7/12 (rplAJL), DNA-directed RNA polymerase subunits beta and beta' (rpoBC), alanyl-tRNA synthetase (alaS), and subunit A of proteinase Clp (clpA). Enzymatic activity and extreme thermostability of purified A. pyrophilus RNA polymerase were verified. Transcription initiation on a DNA construct harboring the T7 A1 promoter was demonstrated by elongation of a 32P-labeled trinucleotide. Phylogenetic analyses of the two largest subunits of bacterial RNA polymerases (beta and beta') showed overall consistency with the 16S rRNA-based phylogeny, except for the positions of the hyperthermophiles A. pyrophilus and Thermotoga maritima and for the location of the root of the domain Bacteria. In the phylogenies for both RNA polymerase subunits beta and beta', A. pyrophilus was placed within the Gram-negative bacteria below the epsilon subdivision of the Proteobacteria. No support was found for the 16S rRNA-based hypothesis that A. pyrophilus might be the deepest branch of the Bacteria, but the cell wall-less mycoplasmas were found with a high confidence at the root of the Bacteria phylogenies. This raised doubts not only about whether the original Bacteria were indeed like the hyperthermophiles, but also concerning the value of single-gene phylogenies for hypotheses about the evolution of organisms.

Expression of lacZ from the promoter of the Escherichia coli spc operon cloned into vectors carrying the W205 trp-lac fusion.

The expression of lacZ has been analyzed and compared in a series of promoter cloning vectors by measuring the amount of lacZ mRNA by hybridization and the amount of beta-galactosidase by standard enzymatic assay. Expression was driven by the promoter, Pspc, of the spc ribosomal protein operon. The vectors contained either the standard W205 trp-lac fusion with the trp operon transcription terminator, trpt, located in the lacZ leader sequence, or a deletion derivative that functionally inactivates trpt. In the presence of trpt, lacZ expression was temperature dependent so that increasing the growth temperature reduced the accumulation of lacZ mRNA and beta-galactosidase activity. The frequency of transcript termination at trpt was estimated to be near zero at 20 degreesC and at about 45% at 37 degreesC. The amount of Pspc-derived lacZ mRNA and the amount of beta-galactosidase produced per lacZ mRNA varied, depending on the mRNA 5' leader sequence between Pspc and lacZ. These results demonstrate that the quantitative assessment of promoter activities with promoter cloning vectors requires careful analysis and interpretation. One particular construct without trpt did not seem to contain fortuitous transcription or translation signals generated at the fusion junction. In this strain, lacZ expression from Pspc was compared at the enzyme activity and mRNA levels with a previously constructed strain in which lacZ was linked to the tandem P1 and P2 promoters of the rrnB operon. At any given growth rate, the different activities of beta-galactosidase in these two strains were found to reflect the same differences in their amounts of lacZ mRNA. Assuming that the promoter-lacZ fusions in these strains reflect the properties of the promoters in their normal chromosomal setting, it was possible to estimate the absolute transcription activity of Pspc and the relative translation efficiency of Pspc-lacZ mRNA at different growth rates. Transcription from the spc promoter was found to increase from about 10 transcripts per min at a growth rate of 1.0 doublings/h to a maximum plateau of about 23 transcripts per min at growth rates above 1.5 doublings/h. The translation frequency of lacZ mRNA expressed from Pspc was unaffected by growth rates.

Transcription analysis of two disparate rRNA operons in the halophilic archaeon Haloarcula marismortui.

The genome of the halophilic archaeon Haloarcula marismortui contains two rRNA operons designated rrnA and rrnB. Genomic clones of the two operons and their flanking regions have been sequenced, and primary transcripts and processing intermediates derived from each operon have been characterized. The 16S, 23S, and 5S genes from the two operons were found to differ at 74 of 1,472 positions, 39 of 2,922 positions, and 2 of 122 positions, respectively. This degree of sequence divergence for multicopy (paralogous) rRNA genes was 10- to 50-fold or more higher than anticipated. The two operons exhibit other profound differences that include (i) the presence in rrnA and the absence in rrnB of tRNAAla and tRNACys genes in the intergenic and distal regions, respectively, (ii) divergent 5' flanking sequences, and (iii) distinct pathways for processing and maturation of 16S rRNA. Processing and maturation of 16S and 23S rRNA from rrnA operon transcripts and of 23S rRNA from rrnB operon transcripts follow the canonical halophilic pathway, whereas maturation of 16S rRNA from rrnB operon transcripts follows an unusual and different pathway that is apparently devoid of any 5' processing intermediate.

Initiation and velocity of chromosome replication in Escherichia coli B/r and K-12.

The macromolecular composition and a number of parameters affecting chromosome replication were examined over a range of exponential growth rates in two common Escherichia coli strains, B/r and K-12 AB1157. Based on improved measurements of DNA after treatment of exponential cultures with rifampin, the cell mass per chromosomal replication origin (initiation mass) and the time required to replicate the chromosome from origin to terminus (C period) were determined. For these two strains, the initiation mass approached values of 8 x 10(-10) and 10 x 10(-10) units of optical density (at 460 nm) of culture mass per oriC, respectively, at growth rates above 1 doubling/h (at 37 degrees C). The amount of protein per oriC decreased with increasing growth rate for AB1157 and remained nearly constant for the B/r strain. The C period decreased for both strains in an essentially identical manner from about 70 min at 0.6 doublings/h to about 33 min at 3 doublings/h. From the initiation mass and C period, relative or absolute copy numbers for genes with known map locations can be accurately determined at different growth rates. At growth rates above 2 doublings/h, when chromosomes are highly branched, genes near the origin are about threefold more prevalent than genes near the terminus. At a growth rate of 0.6 doubling/h, this ratio is only about 1.7, which reflects the lower degree of chromosome branching.

Formation of the 5' end pseudoknot in small subunit ribosomal RNA: involvement of U3-like sequences.

Hughes (1996, J Mol Biol 259:645-654) proposed that the box A region of U3 snoRNA interacts by complementary base pairing with small subunit (ss) rRNA sequences within precursor (pre-) rRNA and through rearrangement and displacement mediates the formation of the universally conserved 5' end pseudoknot. We wondered how this conserved pseudoknot might be formed in the ss rRNAs of archaeal and bacterial organisms that lack a U3 RNA. In examining the 5' external transcribed spacer (5' ETS) region in pre-rRNA transcripts from some of these organisms, we noted the presence of U3 box A-like sequences. By analogy with the U3-ss rRNA intermolecular interaction, we suggest that the box A-like 5' ETS sequence interacts through intramolecular complementary base pairing with the 5' end pseudoknot sequences within pre-rRNA; rearrangement of this structure mediates the formation of the conserved 5' end pseudoknot. If correct, this means that some of the pre-rRNA maturation-folding functions provided in trans by snoRNAs in eukaryotic organisms may be provided in cis by the spacer sequences in pre-rRNA transcripts in some bacterial or archaeal organisms lacking snoRNAs.

Evolutionary divergence and salinity-mediated selection in halophilic archaea.

Halophilic (literally salt-loving) archaea are a highly evolved group of organisms that are uniquely able to survive in and exploit hypersaline environments. In this review, we examine the potential interplay between fluctuations in environmental salinity and the primary sequence and tertiary structure of halophilic proteins. The proteins of halophilic archaea are highly adapted and magnificently engineered to function in an intracellular milieu that is in ionic balance with an external environment containing between 2 and 5 M inorganic salt. To understand the nature of halophilic adaptation and to visualize this interplay, the sequences of genes encoding the L11, L1, L10, and L12 proteins of the large ribosome subunit and Mn/Fe superoxide dismutase proteins from three genera of halophilic archaea have been aligned and analyzed for the presence of synonymous and nonsynonymous nucleotide substitutions. Compared to homologous eubacterial genes, these halophilic genes exhibit an inordinately high proportion of nonsynonymous nucleotide substitutions that result in amino acid replacement in the encoded proteins. More than one-third of the replacements involve acidic amino acid residues. We suggest that fluctuations in environmental salinity provide the driving force for fixation of the excessive number of nonsynonymous substitutions. Tinkering with the number, location, and arrangement of acidic and other amino acid residues influences the fitness (i.e., hydrophobicity, surface hydration, and structural stability) of the halophilic protein. Tinkering is also evident at halophilic protein positions monomorphic or polymorphic for serine; more than one-third of these positions use both the TCN and the AGY serine codons, indicating that there have been multiple nonsynonymous substitutions at these positions. Our model suggests that fluctuating environmental salinity prevents optimization of fitness for many halophilic proteins and helps to explain the unusual evolutionary divergence of their encoding genes.

Conserved sequence elements involved in regulation of ribosomal protein gene expression in halophilic archaea.

A region of the Haloferax volcanii genome encoding ribosomal proteins L11e, L1e, L10e, and L12e was cloned and sequenced, and the transcripts derived from the cluster were characterized. Flanking and noncoding regions of the sequence were analyzed phylogenetically by comparison with the homologous sequences from two other halophilic archaea, i.e., Halobacterium cutirubrum and Haloarcula marismortui. Motifs, identified by high-level sequence conservation, include both transcriptional and translational regulatory elements and other elements of unknown function.

A NusG-like protein from Thermotoga maritima binds to DNA and RNA.

The NusG-like protein from Thermotoga maritima was expressed in Escherichia coli and purified to homogeneity. Purified T. maritima NusG exhibited a generalized, non-sequence-specific and highly cooperative DNA and RNA binding activity. The complexes formed between nucleic acid and T. maritima NusG were unable to penetrate a polyacrylamide or agarose gel. The affinity of the protein for DNA was highest in buffers containing about 50 mM salt. The DNA-protein complexes could not be stained with ethidium bromide, were resistant to digestion by TaqI endonuclease, were able to be transcribed in vitro by T. maritima RNA polymerase, and contained a minimum of about 30 to 40 monomers of NusG per kb of duplex DNA. The protein had comparable affinities for duplex DNA and RNA but a lower affinity for single-stranded DNA. Electron microscopy showed that the DNA in the complex is condensed within a large structure that resembles the complex between DNA and histone-like protein Hcl from Chlamydia trachomatis. Neither the wild-type T. maritima nusG gene nor a deletion derivative more similar to the E. coli gene was able to substitute for the essential E. coli nusG. Two variants of the NusG protein were constructed, expressed, and purified: one contains only the entire 171-amino-acid insertion that is unique to T. maritima NusG, and the other has only the sequences present in NusG homologs from E. coli and other eubacteria. Both variants exhibited similar DNA and RNA binding behavior, although their apparent affinities were 5- to 10-fold lower than that of the wild-type T. maritima NusG.

Preribosomal RNA processing in archaea: characterization of the RNP endonuclease mediated processing of precursor 16S rRNA in the thermoacidophile Sulfolobus acidocaldarius.

The hyperthermophilic archaeon Sulfolobus acidocaldarius uses a novel RNA-containing endonuclease to excise and mature 16S rRNA from the precursor (pre) rRNA transcript. A cell-free processing system has been developed using an in vitro transcribed RNA substrate containing the entire 144 nucleotide 5' external transcribed spacer (5'ETS) and the first 72 nucleotides of 16S rRNA. The cell-free extract cleaves in the 5'ETS at positions -99, -31, and +1 (i.e., the 5'ETS-16S junction). These positions are at or near the positions cleaved in vivo during processing of the pre rRNA transcript. The processing activity has been purified between 100 and 200-fold and appears to contain five or six polypeptide components and perhaps as many as 10 different small RNA components. Using combined reverse transcription-PCR amplification, full or partial cDNA copies of two of the RNA components have been obtained. One of the RNAs exhibits sequence and structural similarities to eukaryotic U3 snoRNA. The processing activity has been shown to be inactivated by micrococcal nuclease. It can be reactivated by reconstituting using bulk RNA from S.acidocaldarius but not bulk RNA from distantly related organisms. The activity is also abolished by RNase H digestion in the presence of oligonucleotides complementary to the U3-like RNA. These results demonstrate that the U3-like RNA is an essential component of the pre rRNA processing RNP endonuclease. Furthermore, this RNP endonuclease is not a derived eukaryotic feature, instead its existence predates the divergence of archaea and eukaryotes.

Ribosomal RNA precursor processing by a eukaryotic U3 small nucleolar RNA-like molecule in an archaeon.

An RNA-containing endonuclease that catalyzes the excision and maturation of the 16S ribosomal RNA (rRNA) from the rRNA primary transcript (pre-rRNA) in the hyperthermophilic archaeon Sulfolobus acidocaldarius has been characterized. The ribonucleoprotein was inactivated by micrococcal nuclease treatment and inactivation was reversed by reconstitution with bulk RNA. A 159-nucleotide RNA with sequence and structural similarity to U3 small nucleolar RNAs of eukaryotes copurified with the endonuclease activity. Oligonucleotide-targeted ribonuclease H inactivation of the U3-like RNA component also abolished processing activity. A motif within the U3 homolog is complementary to the region around the three cleavage sites in the pre-RNA substrate. Thus, U3-mediated processing of pre-rRNA is not specific to eukaryotes; its origin predates the divergence of archaea and eukaryotes.