A novel mutation in the sulfate transporter gene SLC26A2 (DTDST) specific to the Finnish population causes de la Chapelle dysplasia.

BACKGROUND: Mutations in the sulfate transporter gene SLC26A2 (DTDST) cause a continuum of skeletal dysplasia phenotypes that includes achondrogenesis type 1B (ACG1B), atelosteogenesis type 2 (AO2), diastrophic dysplasia (DTD), and recessive multiple epiphyseal dysplasia (rMED). In 1972, de la Chapelle et al reported two siblings with a lethal skeletal dysplasia, which was denoted "neonatal osseous dysplasia" and "de la Chapelle dysplasia" (DLCD). It was suggested that DLCD might be part of the SLC26A2 spectrum of phenotypes, both because of the Finnish origin of the original family and of radiographic similarities to ACG1B and AO2. OBJECTIVE: To test the hypothesis whether SLC26A2 mutations are responsible for DLCD. METHODS: We studied the DNA from the original DLCD family and from seven Finnish DTD patients in whom we had identified only one copy of IVS1+2T>C, the common Finnish mutation. A novel SLC26A2 mutation was found in all subjects, inserted by site-directed mutagenesis in a vector harbouring the SLC26A2 cDNA, and expressed in sulfate transport deficient Chinese hamster ovary (CHO) cells to measure sulfate uptake activity. RESULTS: We identified a hitherto undescribed SLC26A2 mutation, T512K, homozygous in the affected subjects and heterozygous in both parents and in the unaffected sister. T512K was then identified as second pathogenic allele in the seven Finnish DTD subjects. Expression studies confirmed pathogenicity. CONCLUSIONS: DLCD is indeed allelic to the other SLC26A2 disorders. T512K is a second rare "Finnish" mutation that results in DLCD at homozygosity and in DTD when compounded with the milder, common Finnish mutation.

Fine structure of the vaccinia virus gene encoding the precursor of the major core protein 4 a.

A 6008 base pair fragment of the vaccinia virus DNA containing the gene for the precursor of the major core protein 4 a, which has been designated P4 a, was sequenced. A long open reading frame (ORF) encoding a protein of molecular weight 102,157 started close to the position where the P4 a mRNA had been mapped. Analysis of the mRNA by S1 nuclease mapping and primer extension indicated that the 5' end defined by the former method is not the true 5' end. This suggests that the P4 a coding region is preceded by leader sequences that are not derived from the immediate vicinity of the gene, similar to what has been reported for another late vaccinia virus mRNA. The sequenced DNA contained several further ORFs on the same, or opposite DNA strand, providing further evidence for the close spacing of protein-coding sequences in the viral genome.

aIr-1, a newly found locus on mouse chromosome 16 encoding a trans-acting activator factor for MHC class II gene expression.

RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.

Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization.

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C.

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.

DNA sequence of the gene encoding the Zc1 protein from Zea mays W64A

The Zein-2 component named Zc 1 corresponds to a storage protein of an apparent M.W. of 16 kDa present in maize endosperm.

DNA sequence of the gene encoding the Zc1 protein from Zea mays W64A

The Zein-2 component named Zc 1 corresponds to a storage protein of an apparent M.W. of 16 kDa present in maize endosperm.

Low-P tolerance mechanisms and differential gene expression in contrasting wheat genotypes

The objectives of this study were to determine low-P tolerance mechanisms in contrasting wheat genotypes and to evaluate the association of these mechanisms to differential gene expression. Wheat seedlings of cultivars Toropi (tolerant to low-P availability) and Anahuac (sensitive) were evaluated. Seedlings were hydroponically grown in the absence or presence of P (1.0 mmol L-1) during three different time periods: 24, 120 and 240 hours. Free phosphate (Pi) and total P contents were measured in shoots and roots. The experiment's design was in randomized blocks with three replicates, each formed by ten plants. The relative expression of genes encoding the malate transporter TaALMT1 and the transcription factor PTF1 was evaluated. Phosphorus starvation beyond ten days increased the expression of TaALMT1 only in 'Toropi'. PTF1's expression was early induced in both genotypes under P starvation, but remained significant after ten days only in 'Toropi'. Shoot Pi concentration in 'Toropi' was independent from P availability; under starvation, 'Toropi' favored the maintenance of shoot Pi concentration. The low-P tolerance of Toropi cultivar at initial growth stages is mainly due to its ability to maintain constant the Pi shoot level.

Sequencing of Lp-PLA2-encoding PLA2G7 gene in 2000 Europeans reveals several rare loss-of-function mutations.

Elevated plasma levels of lipoprotein-associated phospholipase A(2) (Lp-PLA2) activity have been shown to be associated with increased risk of coronary heart disease and an inhibitor of this enzyme is under development for the treatment of that condition. A Val279Phe null allele in this gene, that may influence patient eligibility for treatment, is relatively common in East Asians but has not been observed in Europeans. We investigated the existence and functional effects of low frequency alleles in a Western European population by re-sequencing the exons of PLA2G7 in 2000 samples. In all, 19 non-synonymous single-nucleotide polymorphisms (nsSNPs) were found, 14 in fewer than four subjects (minor allele frequency <0.1%). Lp-PLA2 activity was significantly lower in rare nsSNP carriers compared with non-carriers (167.8±63.2 vs 204.6±41.8, P=0.01) and seven variants had enzyme activities consistent with a null allele. The cumulative frequency of these null alleles was 0.25%, so <1 in 10,000 Europeans would be expected to be homozygous, and thus not potentially benefit from treatment with an Lp-PLA2 inhibitor.

Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum.

Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in the stratum corneum, nails and hair. In a culture medium containing soy proteins as sole nitrogen source a substantial proteolytic activity was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55-75 % inhibited by o-phenanthroline, attesting that metalloproteases were secreted by all three species. Using a consensus probe constructed on previously characterized genes encoding metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis, a five-member MEP family was isolated from genomic libraries of T. rubrum, T. mentagrophytes and M. canis. A phylogenetic analysis of genomic and protein sequences revealed a robust tree consisting of five main clades, each of them including a MEP sequence type from each dermatophyte species. Each MEP type was remarkably conserved across species (72-97 % amino acid sequence identity). The tree topology clearly indicated that the multiplication of MEP genes in dermatophytes occurred prior to species divergence. In culture medium containing soy proteins as a sole nitrogen source secreted Meps accounted for 19-36 % of total secreted protein extracts; characterization of protein bands by proteolysis and mass spectrometry revealed that the three dermatophyte species secreted two Meps (Mep3 and Mep4) encoded by orthologous genes.

Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens

Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P. fluorescensinsecticidal toxin) that is related to the insect toxin Mcf (Makes caterpillars floppy) of the entomopathogen Photorhabdus luminescens, a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella. In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria

Localization and fine structure of a vaccinia virus gene encoding an envelope antigen.

The major antigen on the envelope of extracellular vaccinia virus particles is a polypeptide with an apparent molecular weight of 37,000 (p37K; G. Hiller and K. Weber, J. Virol. 55:651-659, 1985). The gene encoding p37K was mapped in the vaccinia virus genome by hybrid selection of RNA followed by in vitro translation. p37K was then identified among the in vitro translation products by immunoprecipitation with a monoclonal antibody. The gene is located close to the right-hand end of the HindIII F fragment. The corresponding region of the DNA was sequenced, and an open reading frame encoding a polypeptide of 41,748 daltons was observed. The 5' end of the mRNA, as defined by nuclease S1 analysis, maps within only a few nucleotides of the translation initiation codon. Examination of the DNA sequence around the putative initiation site of transcription revealed a characteristic sequence, TAAATG, which includes the ATG translation initiation codon and which is conserved in all but one late gene so far analyzed. It is therefore likely that this sequence is an important regulatory signal for late gene expression in vaccinia virus.

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.

Enhanced anti-tumor effect of a gene gun-delivered DNA vaccine encoding the human papillomavirus type 16 oncoproteins genetically fused to the herpes simplex virus glycoprotein D

Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70% of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50% of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.

The γ-tocopherol-like family of N-methyltransferases: A taxonomically clustered gene family encoding enzymes responsible for N-methylation of monoterpene indole alkaloids

The plant family Apocynaceae accumulates thousands of monoterpene indole alkaloids (MIAs) which originate, biosynthetically, from the common secoiridoid intermediate, strictosidine, that is formed from the condensation of tryptophan and secologanin molecules. MIAs demonstrate remarkable structural diversity and have pharmaceutically valuable biological activities. For example; a subunit of the potent anti-neoplastic molecules vincristine and vinblastine is the aspidosperma alkaloid, vindoline. Vindoline accumulates to trace levels under natural conditions. Research programs have determined that there is significant developmental and light regulation involved in the biosynthesis of this MIA. Furthermore, the biosynthetic pathway leading to vindoline is split among at least five independent cell types. Little is known of how intermediates are shuttled between these cell types. The late stage events in vindoline biosynthesis involve six enzymatic steps from tabersonine. The fourth biochemical step, in this pathway, is an indole N-methylation performed by a recently identified N-methyltransfearse (NMT). For almost twenty years the gene encoding this NMT had eluded discovery; however, in 2010 Liscombe et al. reported the identification of a γ-tocopherol C-methyltransferase homologue capable of indole N-methylating 2,3-dihydrotabersonine and Virus Induced Gene Silencing (VIGS) suppression of the messenger has since proven its involvement in vindoline biosynthesis. Recent large scale sequencing initiatives, performed on non-model medicinal plant transcriptomes, has permitted identification of candidate genes, presumably involved, in MIA biosynthesis never seen before in plant specialized metabolism research. Probing the transcriptome assemblies of Catharanthus roseus (L.)G.Don, Vinca minor L., Rauwolfia serpentine (L.)Benth ex Kurz, Tabernaemontana elegans, and Amsonia hubrichtii, with the nucleotide sequence of the N-methyltransferase involved in vindoline biosynthesis, revealed eight new homologous methyltransferases. This thesis describes the identification, molecular cloning, recombinant expression and biochemical characterization of two picrinine NMTs, one from V. minor and one from R. serpentina, a perivine NMT from C. roseus, and an ajmaline NMT from R. serpentina. While these TLMTs were expressed and functional in planta, they were active at relatively low levels and their N-methylated alkaloid products were not apparent our from alkaloid isolates of the plants. It appears that, for the most part, these TLMTs, participate in apparently silent biochemical pathways, awaiting the appropriate developmental and environmental cues for activity.