Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

Histological analyses of the fungal endophytes in Rosa hybrida

Endophytes are fungi that cause asymptomatic infections in leaves and stems of healthy plants. This study evaluated the presence of endophytic fungi on leaves of Rosa hybrida collected in Bogota, (Colombia) using histological techniques and light microscopy. Histological preparations showed fungal mycelium with few cells in the vascular tissue. Colonization of mesophyll cells was not observed. Visualization of fungal cells within plant tissues is a confirmatory test of the endophytic habitat of fungi isolated from leaves of Rosa hybrida.     

Molecular Typing of Lung Adenocarcinoma on Cytological Samples Using a Multigene Next Generation Sequencing Panel

Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%). In 24/36 cases (67%) at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16%) and 10/36 (28%) cases, were mutually exclusive. Nine samples (25%) showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy.     

Isolation of cDNAs and functional characterisation of two multi-product terpene synthase enzymes from sandalwood, Santalum album L

Sandalwood, Santalum album (Santalaceae) is a small hemi-parasitic tropical tree of great economic value. Sandalwood timber contains resins and essential oils, particularly the santalols, santalenes and dozens of other minor sesquiterpenoids. These sesquiterpenoids provide the unique sandalwood fragrance. The research described in this paper set out to identify genes involved in essential oil biosynthesis, particularly terpene synthases (TPS) in S. album, with the long-term aim of better understanding heartwood oil production. Degenerate TPS primers amplified two genomic TPS fragments from S. album, one of which enabled the isolation of two TPS cDNAs, SamonoTPS1 (1731 bp) and SasesquiTPS1 (1680 bp). Both translated protein sequences shared highest similarity with known TPS from grapevine (Vitis vinifera). Heterologous expression in Escherichia coli produced catalytically active proteins. SamonoTPS1 was identified as a monoterpene synthase which produced a mixture of (+)-α-terpineol and (−)-limonene, along with small quantities of linalool, myrcene, (−)-α-pinene, (+)-sabinene and geraniol when assayed with geranyl diphosphate. Sesquiterpene synthase SasesquiTPS1 produced the monocyclic sesquiterpene alcohol germacrene D-4-ol and helminthogermacrene, when incubated with farnesyl diphosphate. Also present were α-bulnesene, γ-muurolene, α- and β-selinenes, as well as several other minor bicyclic compounds. Although these sesquiterpenes are present in only minute quantities in the distilled sandalwood oil, the genes and their encoded enzymes described here represent the first TPS isolated and characterised from a member of the Santalaceae plant family and they may enable the future discovery of additional TPS genes in sandalwood.     

NO role of NOS2A susceptibility polymorphisms in rheumatoid arthritis

Nitric oxide has been described as a trigger for the synthesis of proinflammatory mediators and as a cytotoxic molecule with a pivotal role in apoptosis at the joints of rheumatoid arthritis (RA) patients. Polymorphisms in the NOS2A gene, which codes for the inducible nitric oxide synthase [(i)NOS], have been tested for association with several autoimmune diseases such as Crohn’s disease or type 1 diabetes. Moreover, the existence of correlated levels of (i)NOS protein and synovial cell apoptosis in RA patients, pointed to NOS2A as a good candidate gene involved in RA predisposition. The role of NOS2A was studied in 405 Spanish RA patients and in 398 ethnically matched healthy controls, through the analysis of five SNPs: two at the NOS2A promoter (rs2779251 and 2779248), other two exonic markers (Asp³⁴⁶Asp (rs1137933) and Ser⁶⁰⁸Leu (rs22518)) and the last one located at intron 7 (rs3729508). We also included other two widely-used promoter polymorphisms: the insertion/deletion (TAAA/-) and the (CCTTT)n microsatellite. No individual association of each single-marker or haplotype was found with RA susceptibility. Our data show the low linkage disequilibrium between these NOS2A SNPs and the alleles of the (CCTTT)n microsatellite, corroborating in a Spanish population the observation previously described in British and Gambian population. The present data do not support a causative role of NOS2A polymorphisms in RA predisposition.     

Phylogenetic ecology of widespread uncultured clades of the Kingdom Euryarchaeota

Despite its widespread distribution and high levels of phylogenetic diversity, microbes are poorly understood creatures. We applied a phylogenetic ecology approach in the Kingdom Euryarchaeota (Archaea) to gain insight into the environmental distribution and evolutionary history of one of the most ubiquitous and largely unknown microbial groups. We compiled 16S rRNA gene sequences from our own sequence libraries and public genetic databases for two of the most widespread mesophilic Euryarchaeota clades, Lake Dagow Sediment (LDS) and Rice Cluster-V (RC-V). The inferred population history indicated that both groups have undergone specific nonrandom evolution within environments, with several noteworthy habitat transition events. Remarkably, the LDS and RC-V groups had enormous levels of genetic diversity when compared with other microbial groups, and proliferation of sequences within each single clade was accompanied by significant ecological differentiation. Additionally, the freshwater Euryarchaeota counterparts unexpectedly showed high phylogenetic diversity, possibly promoted by their environmental adaptability and the heterogeneous nature of freshwater ecosystems. The temporal phylogenetic diversification pattern of these freshwater Euryarchaeota was concentrated both in early times and recently, similarly to other much less diverse but deeply sampled archaeal groups, further stressing that their genetic diversity is a function of environment plasticity. For the vast majority of living beings on Earth (i.e. the uncultured microorganisms), how they differ in the genetic or physiological traits used to exploit the environmental resources is largely unknown. Inferring population history from 16S rRNA gene-based molecular phylogenies under an ecological perspective may shed light on the intriguing relationships between lineage, environment, evolution and diversity in the microbial world.     

Amplification and Phylogenetic Relationships of a Subfamily of blood, a Retrotransposable Element of Drosophila

To get a better understanding of the effect of interelement selection on the variation of long terminal repeat retrotransposon families, we have investigated the evolutionary history of blood in the Drosophila melanogaster species complex. We carried out a PCR approach to amplify the 5′ untranslated region from blood in the four species of the complex. This procedure revealed two main classes of size variants. Phylogenetic analyses of nucleotide sequences from these variants and blood elements from the Drosophila Genome Projects database show that elements are grouped according to their size, so that they probably correspond to two subfamilies. These two subfamilies arose prior to the split of the complex, and several facts indicate that the expansion of one of them is leading to the competitive exclusion of the other, at least from the euchromatic regions of the genome. Received: 17 August 2000 / Accepted: 20 November 2000     

Free energy surfaces of beta-hairpin and alpha-helical peptides generated by replica exchange molecular dynamics with the AGBNP implicit solvent model

We have studied the potential of mean force of two peptides, one known to adopt a beta-hairpin and the other an alpha-helical conformation in solution. These peptides are, respectively, residues 41-56 of the C-terminus (GEWTYDDATKTFTVTE) of the B1 domain of protein G and the 13 residue C-peptide (KETAAAKFERQHM) of ribonuclease A. Extensive canonical ensemble sampling has been performed using a parallel replica exchange method. The effective potential employed in this work consists of the OPLS all-atom force field (OPLS-AA) and an analytical generalized Born (AGB) implicit solvent model including a novel nonpolar solvation free energy estimator (NP). An additional dielectric screening parameter has been incorporated into the AGBNP model. In the case of the beta-hairpin, the nonpolar solvation free energy estimator provides the necessary effective interactions for the collapse of the hydrophobic core (W43, Y45, F52, and V54), which the more commonly used surface-area-dependent nonpolar model does not provide. For both the beta-hairpin and the alpha-helix, increased dielectric screening reduces the stability of incorrectly formed salt bridges, which tend to disrupt the formation of the hairpin and helix, respectively. The fraction of beta-hairpin and alpha-helix content we obtained using the AGBNP model agrees well with experimental results. The thermodynamic stability of the beta-hairpin from protein G and the alpha-helical C-peptide from ribonuclease A as modeled with the OPLS-AA/AGBNP effective potential reflects the balance between the nonpolar effective potential terms, which drive compaction, and the polar and hydrogen bonding terms, which promote secondary structure formation.     

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3(-/-) mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3(-/-) mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-alpha-trypsin inhibitor is normal in Ptx3(-/-) cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor alpha-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.