The coupon collector and the suppressor mutation: estimating the number of compensatory mutations by maximum likelihood

Compensatory mutation occurs when a loss of fitness caused by a deleterious mutation is restored by its epistatic interaction with a second mutation at a different site in the genome. How many different compensatory mutations can act on a given deleterious mutation? Although this quantity is fundamentally important to understanding the evolutionary consequence of mutation and the genetic complexity of adaptation, it remains poorly understood. To determine the shape of the statistical distribution for the number of compensatory mutations per deleterious mutation, we have performed a maximum-likelihood analysis of experimental data collected from the suppressor mutation literature. Suppressor mutations are used widely to assess protein interactions and are under certain conditions equivalent to compensatory mutations. By comparing the maximum likelihood of a variety of candidate distribution functions, we established that an L-shaped gamma distribution (alpha=0.564, theta=21.01) is the most successful at explaining the collected data. This distribution predicts an average of 11.8 compensatory mutations per deleterious mutation. Furthermore, the success of the L-shaped gamma distribution is robust to variation in mutation rates among sites. We have detected significant differences among viral, prokaryotic, and eukaryotic data subsets in the number of compensatory mutations and also in the proportion of compensatory mutations that are intragenic. This is the first attempt to characterize the overall diversity of compensatory mutations, identifying a consistent and accurate prior distribution of compensatory mutation diversity for theoretical evolutionary models.     

Solution structure of Kti11p from Saccharomyces cerevisiae reveals a novel zinc-binding module

Kti11p is a small, highly conserved CSL zinc finger-containing protein found in many eukaryotes. It was first identified as one of the factors required for maintaining the sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin. Then, it was found to be identical to Dph3, a protein required for diphthamide biosynthesis on eEF-2, the target of diphtheria toxin and Pseudomonas exotoxin A, in both yeast and higher eukaryotes. Furthermore, Kti11p/Dph3 was found to physically interact with core-Elongator, ribosomal proteins, eEF-2, two other proteins required for diphthamide modification on eEF-2, and DelGEF. Here, we determined the solution structure of Kti11p using NMR, providing the first structure of the CSL-class zinc-binding protein family. We present the first experimental evidence that Kti11p can bind a single Zn(2+) ion by its four conserved cysteine residues. The major structure of Kti11p comprises a beta sandwich as well as an alpha helix. Moreover, a structure-based similarity search suggests that it represents a novel structure and may define a new family of the zinc ribbon fold group. Therefore, our work provides a molecular basis for further understanding the multiple functions of Kti11p/Dph3 in different biological processes.     

Chemical xenobiotics and mitochondrial autoantigens in primary biliary cirrhosis: identification of antibodies against a common environmental, cosmetic, and food additive, 2-octynoic acid

Emerging evidence has suggested environmental factors as causative agents in the pathogenesis of primary biliary cirrhosis (PBC). We have hypothesized that in PBC the lipoyl domain of the immunodominant E2 component of pyruvate dehydrogenase (PDC-E2) is replaced by a chemical xenobiotic mimic, which is sufficient to break self-tolerance. To address this hypothesis, based upon our quantitative structure-activity relationship data, a total of 107 potential xenobiotic mimics were coupled to the lysine residue of the immunodominant 15 amino acid peptide of the PDC-E2 inner lipoyl domain and spotted on microarray slides. Sera from patients with PBC (n = 47), primary sclerosing cholangitis (n = 15), and healthy volunteers (n = 20) were assayed for Ig reactivity. PBC sera were subsequently absorbed with native lipoylated PDC-E2 peptide or a xenobiotically modified PDC-E2 peptide, and the remaining reactivity analyzed. Of the 107 xenobiotics, 33 had a significantly higher IgG reactivity against PBC sera compared with control sera. In addition, 9 of those 33 compounds were more reactive than the native lipoylated peptide. Following absorption, 8 of the 9 compounds demonstrated cross-reactivity with lipoic acid. One compound, 2-octynoic acid, was unique in both its quantitative structure-activity relationship analysis and reactivity. PBC patient sera demonstrated high Ig reactivity against 2-octynoic acid-PDC-E2 peptide. Not only does 2-octynoic acid have the potential to modify PDC-E2 in vivo but importantly it was/is widely used in the environment including perfumes, lipstick, and many common food flavorings.     

A 2500-locus bin map of wheat homoeologous group 5 provides insights on gene distribution and colinearity with rice

We constructed high-density deletion bin maps of wheat chromosomes 5A, 5B, and 5D, including 2338 loci mapped with 1052 EST probes and 217 previously mapped loci (total 2555 loci). This information was combined to construct a consensus chromosome bin map of group 5 including 24 bins. A relatively higher number of loci were mapped on chromosome 5B (38%) compared to 5A (34%) and 5D (28%). Differences in the levels of polymorphism among the three chromosomes were partially responsible for these differences. A higher number of duplicated loci was found on chromosome 5B (42%). Three times more loci were mapped on the long arms than on the short arms, and a significantly higher number of probes, loci, and duplicated loci were mapped on the distal halves than on the proximal halves of the chromosome arms. Good overall colinearity was observed among the three homoeologous group 5 chromosomes, except for the previously known 5AL/4AL translocation and a putative small pericentric inversion in chromosome 5A. Statistically significant colinearity was observed between low-copy-number ESTs from wheat homoeologous group 5 and rice chromosomes 12 (88 ESTs), 9 (72 ESTs), and 3 (84 ESTs).     

A 2600-locus chromosome bin map of wheat homoeologous group 2 reveals interstitial gene-rich islands and colinearity with rice

The complex hexaploid wheat genome offers many challenges for genomics research. Expressed sequence tags facilitate the analysis of gene-coding regions and provide a rich source of molecular markers for mapping and comparison with model organisms. The objectives of this study were to construct a high-density EST chromosome bin map of wheat homoeologous group 2 chromosomes to determine the distribution of ESTs, construct a consensus map of group 2 ESTs, investigate synteny, examine patterns of duplication, and assess the colinearity with rice of ESTs assigned to the group 2 consensus bin map. A total of 2600 loci generated from 1110 ESTs were mapped to group 2 chromosomes by Southern hybridization onto wheat aneuploid chromosome and deletion stocks. A consensus map was constructed of 552 ESTs mapping to more than one group 2 chromosome. Regions of high gene density in distal bins and low gene density in proximal bins were found. Two interstitial gene-rich islands flanked by relatively gene-poor regions on both the short and long arms and having good synteny with rice were discovered. The map locations of two ESTs indicated the possible presence of a small pericentric inversion on chromosome 2B. Wheat chromosome group 2 was shown to share syntenous blocks with rice chromosomes 4 and 7.     

Deletion mapping of homoeologous group 6-specific wheat expressed sequence tags

To localize wheat (Triticum aestivum L.) ESTs on chromosomes, 882 homoeologous group 6-specific ESTs were identified by physically mapping 7965 singletons from 37 cDNA libraries on 146 chromosome, arm, and sub-arm aneuploid and deletion stocks. The 882 ESTs were physically mapped to 25 regions (bins) flanked by 23 deletion breakpoints. Of the 5154 restriction fragments detected by 882 ESTs, 2043 (loci) were localized to group 6 chromosomes and 806 were mapped on other chromosome groups. The number of loci mapped was greatest on chromosome 6B and least on 6D. The 264 ESTs that detected orthologous loci on all three homoeologs using one restriction enzyme were used to construct a consensus physical map. The physical distribution of ESTs was uneven on chromosomes with a tendency toward higher densities in the distal halves of chromosome arms. About 43% of the wheat group 6 ESTs identified rice homologs upon comparisons of genome sequences. Fifty-eight percent of these ESTs were present on rice chromosome 2 and the remaining were on other rice chromosomes. Even within the group 6 bins, rice chromosomal blocks identified by 1-6 wheat ESTs were homologous to up to 11 rice chromosomes. These rice-block contigs were used to resolve the order of wheat ESTs within each bin.     

Development of an expressed sequence tag (EST) resource for wheat (Triticum aestivum L.): EST generation, unigene analysis, probe selection and bioinformatics for a 16,000-locus bin-delineated map

This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5′ and 3′ sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics.     

In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.     

Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB

ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (K(m)) of 104.9 +/- 5.4 microm and 90.1 +/- 3.7 microm, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H(+) gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1:1.     

Down-regulation of survivin in nitric oxide-induced cell growth inhibition and apoptosis of the human lung carcinoma cells

Survivin is expressed in most tumor cells and has been associated with both anti-apoptosis and mitotic progression. However, the mechanism of regulation of the survivin expression remains unclear. In this study we investigated the expression and regulation of survivin in the nitric oxide (NO)-exposed human lung carcinoma cells. The lung carcinoma cell lines CL3, H1299, and A549 but not normal lung fibroblast expressed high levels of survivin proteins. NO donors S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) decreased the survivin expression. SNAP (0.4 mm, 24h)and SNP (1 mm, 24 h) significantly induced cytotoxicity and apoptosis in lung carcinoma cells. Furthermore, SNAP inhibited the cell growth and increased the fractions of G(2)/M phase. The levels of cyclin B1 and phospho-cdc2-(Thr-161) proteins were inhibited in the NO-exposed cells. The cdc25 phosphatase inhibitors (Cpd 5 and NSC 663284) and the cdc2 kinase inhibitors (alsterpaullone and purvalanol A) enhanced SNP-induced cytotoxicity and the decrease in survivin expression. However, overexpression of survivin by a pOTB7-survivin vector reduced SNP-induced cell growth inhibition and cytotoxicity. In addition, SNP activated the phosphorylation of p38 mitogen-activated protein (MAP) kinase. The specific p38 MAP kinase inhibitor, SB202190, significantly decreased the cytotoxicity and increased the survivin levels in NO donor-treated and inducible NOS-transfected cells. Conversely, anticancer agents including quercetin, arsenite, and cisplatin but not genistein increased the levels of survivin protein. Our results indicated for the first time that NO inhibited the expression of survivin, which was down-regulated by the p38 MAP kinase pathway.