Single nucleotide polymorphism genotyping using short,fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection

Locked nucleic acids (LNAs) are synthetic nucleic acid analogs that bind to complementary target molecules (DNA, RNA or LNA) with very high affinity. At the same time, this binding affinity is decreased substantially when the hybrids thus formed contain even a single mismatched base pair. We have exploited these properties of LNA probes to develop a new method for single nucleotide polymorphism genotyping. In this method, very short (hexamer or heptamer) LNA probes are labeled with either rhodamine or hexachlorofluorescein (HEX), and their hybridization to target DNAs is followed by measuring the fluorescence polarization (FP) of the dyes. The formation of perfectly complementary double-stranded hybrids gives rise to significant FP increases, whereas the presence of single mismatches results in very small or no changes of this parameter. Multiplexing of the assay can be achieved by using differentially labeled wild-type and mutant specific probes in the same solution. The method is homogeneous, and because of the use of extremely short LNA probes, the generation of a universal set of genotyping reagents is possible.     

8-(Hydroxymethyl)-3,N(4)-etheno-C, a potential carcinogenic glycidaldehyde product, miscodes in vitro using mammalian DNA polymerases

8-(Hydroxymethyl)-3,N(4)-etheno-C (8-HM-epsilonC) is an exocyclic adduct resulting from the reaction of dC with glycidaldehyde, a mutagen and animal carcinogen. This compound has now been synthesized and its phosphoramidite incorporated site-specifically into a defined 25-mer oligonucleotide. In this study, the mutagenic potential of this adduct in the 25-mer oligonucleotide was investigated in an in vitro primer-template extension assay using four mammalian DNA polymerases. The miscoding potentials were also compared to those of an analogous derivative, 3,N(4)-etheno C (epsilonC), in the same sequence. Both adducts primarily blocked replication by calf thymus DNA polymerase alpha at the modified base, while human polymerase beta catalyzed measurable replication synthesis through both adducts. Nucleotide insertion experiments showed that dA and dC were incorporated by pol beta opposite either adduct, which would result in a C --> T transition or C --> G transversion. Human polymerase eta, a product of the xeroderma pigmentosum variant (XP-V) gene, catalyzed the most efficient bypass of the two lesions with 25% and 32% for 8-HM-epsilonC and epsilonC bypassed after 15 min. Varying amounts of all four bases opposite the modified bases resulted with pol eta. Human polymerase kappa primarily blocked synthesis at the base prior to the adduct. However, some specific misincorporation of dT resulted, forming an epsilonC.T or 8-HM-epsilonC.T pair. From these data, we conclude that the newly synthesized glycidaldehyde-derived adduct, 8-HM-epsilonC, is a miscoding lesion. The bypass efficiency and insertion specificity of 8-HM-epsilonC and epsilonC were similar for all four polymerases tested, which could be attributed to the similar planarity and sugar conformations for these two derivatives as demonstrated by molecular modeling studies.     

Functional expression of murine LRP1 requires correction of Lrp1 cDNA sequences

Here, we describe the reconstruction of a functional 14 kbp full-length murine Lrp1 cDNA from overlapping partial cDNAs, which were described before [Biochim. Biophys. Acta 1173 (1993) 71]. The reconstructed full-length cDNA needed sequence correction (by mutagenesis) due to nucleotide errors present in the underlying partial cDNAs. These mistakes compromised the proteolytical maturation of the LRP precursor (4545 aa) into its alpha- and beta-subunits. To identify these mistakes initially, detailed sequence analyses and comparison of genomic and cDNA sequences of different murine strains proved to be necessary to obtain correct wild-type sequences. Comparison of Lrp1 cDNA sequences of CBA mice with Lrp1 genomic exon sequences of 129P3/J mice (like in man 89 exons) revealed only 24 nucleotide differences within about 14.8 kbp. Only 1 out of 23 nucleotide differences in the protein coding region affected an amino acid residue: Thr versus Ala at amino acid residue position 2642 in 129P3/J and CBA, respectively. After correction by mutagenesis, both a 129P3/J and a CBA-based version of a full-length wild-type Lrp1 cDNA were functionally expressed in an LRP-deficient mutant CHO cell line. Transient expression showed the expected maturation of the LRP precursor into its two subunits. Furthermore, stable transfection restored the sensitivity to exposure to Pseudomonas aeruginosa toxin A (PEA). Since LRP is the unique receptor for this toxin, this indicates that the toxin could enter the cells after binding to and endocytosis by its genuine receptor. This murine LRP expression system will be instrumental in future experimental dissection of this multifunctional receptor.     

Molecular diversity among marine picophytoplankton as revealed by psbA analyses

Photosynthetic microorganisms play a crucial role in the marine environment. In vast areas of the oceans, marine primary productivity is performed by cells smaller than 2-3 micro m (picoplankton). Here, we report on molecular analyses of the conserved photosynthetic psbA gene (coding for protein D1 of photosystem II reaction centre) as a diversity indicator of naturally occurring marine oxygenic picophytoplankton. The psbA genes proved to be good indicators of the presence of a wide variety of photosynthetic marine microbial groups, including new cyanobacterial groups and eukaryotic algae (prasinophytes). Furthermore, using environmental bacterial artificial chromosome (BAC) libraries, we were able to correlate psbA genes with small subunit rRNAs and, therefore, to confirm their phylogenetic affiliation.     

Evolutionary dynamics of oncogenes and tumor suppressor genes: higher intensities of purifying selection than other genes

Oncogenes and tumor suppressor genes (hereafter referred to as "cancer genes") result in cancer when they experience substitutions that prevent or distort their normal function. We examined evolutionary pressures acting on cancer genes and other classes of disease-related genes and compared our results to analyses of genes without known association to disease. We compared synonymous and nonsynonymous substitution rates in 3,035 human genes-approximately 10% of the genome-measuring the intensity of purifying selection on 311 human disease genes, including 122 cancer-related genes. Although the genes examined are similar to nondisease genes in product, expression, function, and pathway affiliation, we found intriguing differences in the selective pressures experienced by cancer genes relative to other (noncancer) disease-related and non-disease-related genes. We found a statistically significant increase in the intensity of purifying selection exerted on cancer genes (the average ratio of nonsynonymous to synonymous substitutions, omega, was 0.079) relative to all other disease-related genes groups (omega = 0.101) and non-disease-related genes (omega = 0.100). This difference indicates a striking increase in selection against nonsynonymous substitutions in oncogenes and tumor suppressor genes. This finding provides insight into the etiology of cancer and the differences between genes involved in cancer and those implicated in other human diseases. Specifically, we found a significant overlap between human oncogenes and tumor suppressor genes and "essential genes," human homologs of mouse lethal genes identified by knockout experiments. This insight may improve our ability to identify cancer-related genes and enhances our understanding of the nature of these genes.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> glucosyltransferases with activities toward both endogenous and xenobiotic substrates

Arabidopsis thaliana Heynh. harbors UDP-glucose-dependent glucosyltransferase (UGT; EC 2.4.1.-) activities that are able to glucosylate xenobiotic substrates as a crucial step in their detoxification, similar to other plants. However, it has remained elusive whether side-activities of UGTs acting on endogenous substrates could account for that property. Therefore, seven recombinantly expressed A. thaliana enzymes were tested using the phytotoxic xenobiotic model compound 2,4,5-trichlorophenol (TCP) as a substrate. The enzymes were selected from the large Arabidopsis UGT gene family because their previously identified putative endogenous substrates comprised both carboxylic acid, and phenolic and aliphatic hydroxyl moieties as biochemical targets. In addition, UGT75D1, which was shown to accept the endogenous flavonoid kaempferol as a substrate, was included. All enzymes tested, except the sterol-conjugating UGT80A2, glucosylated TCP as a parallel activity. The K(m) values for TCP ranged from 0.059 to 1.25 mM. When tested at saturating concentrations of the native substrates the glucosylation of TCP by the glucose-ester-forming UGT84A1 and UGT84A2 was suppressed by p-coumaric acid and sinapic acid, respectively. In contrast, the activities of UGT72E2 and UGT75D1 toward their phenolic native substrates and the xenobiotic TCP were mutually inhibited. TCP was a competitive inhibitor of sinapyl alcohol glucosylation by UGT72E2. These overlapping in vitro activities suggest cross-talk between the detoxification of xenobiotics and endogenous metabolism at the biochemical level, depending on the presence of competing substrates and enzymes.     

Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: preserved folding energy drives fiber formation

Most gram-negative pathogens express fibrous adhesive virulence organelles that mediate targeting to the sites of infection. The F1 capsular antigen from the plague pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and serves as a prototype for nonpilus organelles assembled via the chaperone/usher pathway. Genetic data together with high-resolution X-ray structures corresponding to snapshots of the assembly process reveal the structural basis of fiber formation. Comparison of chaperone bound Caf1 subunit with the subunit in the fiber reveals a novel type of conformational change involving the entire hydrophobic core of the protein. The observed conformational change suggests that the chaperone traps a high-energy folding intermediate of Caf1. A model is proposed in which release of the subunit allows folding to be completed, driving fiber formation.     

A Synechococcus PglnA::luxAB fusion for estimation of nitrogen bioavailability to freshwater cyanobacteria

In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations. This situation may be due partly to the complexity in estimating its utilization and bioavailability. In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, P glnA, from Synechococcus sp. strain PCC7942 to the luxAB luciferase-encoding genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus chromosome to yield the reporter strain GSL. Light emission by this strain was dependent upon ambient nitrogen concentrations. The linear response range of the emitted luminescence was 1 mM to 1 micro M for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea. When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen.     

Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.