Isolation of Schizosaccharomyces pombe isopentenyl diphosphate isomerase cDNA clones by complementation and synthesis of the enzyme in Escherichia coli

Journal ArticleIsopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprene biosynthetic pathway. The Saccharomyces cerevisiae gene for IPP isomerase, IDI1, was recently isolated and characterized (Anderson, M. S., Muehlbacher, M., Street, I. P., Proffitt, J., and Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175), and the wild-type gene, IDI1, was disrupted with a LEU2 marker to create a diploid yeast strain heterozygous for the idi1::leu2 disruption, which revealed that IDI1 was an essential single-copy gene (Mayer, M.P., Hahn, F. M., Stillman, D. J., and Poulter, C. D. (1992) Yeast 8, 743-748). We now report the isolation of a cDNA clone from Schizosaccharomyces pombe by a plasmid shuffle-mediated complementation of the LEU2 disrupted yeast gene. The S. pombe clone encoded a 26,864-dalton polypeptide of 227 amino acids with a high degree of similarity to the S. cerevisiae IDI1 enzyme. S. pombe IPP isomerase contained the essential Cys and Glu catalytic residues identified in yeast isomerase (Street, I. P., Coffman, H. R., Baker, J., and Poulter, C. (1994) Biochemistry 33, 4212-4217) but was significantly smaller than the S. cerevisiae enzyme. The plasmid shuffle technique is an excellent procedure for screening expression libraries for IPP isomerase activity by complementation of the idi1 mutation

Comparative analysis of the complete sequence of the plastid genome of Parthenium argentatum and identification of DNA barcodes to differentiate Parthenium species and lines

<p>Abstract</p> <p>Background</p> <p><it>Parthenium argentatum </it>(guayule) is an industrial crop that produces latex, which was recently commercialized as a source of latex rubber safe for people with Type I latex allergy. The complete plastid genome of <it>P. argentatum </it>was sequenced. The sequence provides important information useful for genetic engineering strategies. Comparison to the sequences of plastid genomes from three other members of the Asteraceae, <it>Lactuca sativa, Guitozia abyssinica </it>and <it>Helianthus annuus </it>revealed details of the evolution of the four genomes. Chloroplast-specific DNA barcodes were developed for identification of <it>Parthenium </it>species and lines.</p> <p>Results</p> <p>The complete plastid genome of <it>P. argentatum </it>is 152,803 bp. Based on the overall comparison of individual protein coding genes with those in <it>L. sativa, G. abyssinica </it>and <it>H. annuus</it>, we demonstrate that the <it>P. argentatum </it>chloroplast genome sequence is most closely related to that of <it>H. annuus</it>. Similar to chloroplast genomes in <it>G. abyssinica, L. sativa </it>and <it>H. annuus</it>, the plastid genome of <it>P. argentatum </it>has a large 23 kb inversion with a smaller 3.4 kb inversion, within the large inversion. Using the <it>mat</it>K and <it>psb</it>A-<it>trn</it>H spacer chloroplast DNA barcodes, three of the four <it>Parthenium </it>species tested, <it>P. tomentosum</it>, <it>P. hysterophorus </it>and P. <it>schottii</it>, can be differentiated from <it>P. argentatum</it>. In addition, we identified lines within <it>P. argentatum</it>.</p> <p>Conclusion</p> <p>The genome sequence of the <it>P. argentatum </it>chloroplast will enrich the sequence resources of plastid genomes in commercial crops. The availability of the complete plastid genome sequence may facilitate transformation efficiency by using the precise sequence of endogenous flanking sequences and regulatory elements in chloroplast transformation vectors. The DNA barcoding study forms the foundation for genetic identification of commercially significant lines of <it>P</it>. <it>argentatum </it>that are important for producing latex.</p

Remodeling the Isoprenoid Pathway in Tobacco by Expressing the Cytoplasmic Mevalonate Pathway in Chloroplasts

Metabolic engineering to enhance production of isoprenoid metabolites for industrial and medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is produced by the mevalonate pathway (MEV) in the cytosol and by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. A multi-gene approach was employed to insert the entire cytosolic MEV pathway into the tobacco chloroplast genome. Molecular analysis confirmed the site-specific insertion of seven transgenes and homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin, which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV pathway genes accumulated higher levels of mevalonate, carotenoids, squalene, sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic pathway with six enzymes has been transplastomically expressed in plants. Thus, we have developed an important tool to redirect metabolic fluxes in the isoprenoid biosynthesis pathway and a viable multigene strategy for engineering metabolism in plants

Gene Loss and Adaptation to Hominids Underlie the Ancient Origin of HIV-1

SummaryHIV-1 resulted from cross-species transmission of SIVcpz, a simian immunodeficiency virus that naturally infects chimpanzees. SIVcpz, in turn, is a recombinant between two SIV lineages from Old World monkeys. Lentiviral interspecies transmissions are partly driven by the evolution and capacity of viral accessory genes, such as vpx, vpr, and vif, to antagonize host antiviral factors, such as SAMHD1 and the APOBEC3 proteins. We show that vpx, which in other lentiviruses antagonizes SAMHD1, was deleted during the creation of SIVcpz. This genomic deletion resulted in the reconstruction of the overlapping vif gene by “overprinting,” creating a unique vif that overlaps in its 3′ end with the vpr gene and can antagonize hominid APOBEC3s. Moreover, passage of SIVs through chimpanzees facilitated the subsequent adaptation of HIV-1 to humans. Thus, HIV-1 originated through a series of gene loss and adaptation events that generated its chimpanzee precursor and lowered the species barrier to human infection

Strained tetragonal states and Bain paths in metals

Paths of tetragonal states between two phases of a material, such as bcc and fcc, are called Bain paths. Two simple Bain paths can be defined in terms of special imposed stresses, one of which applies directly to strained epitaxial films. Each path goes far into the range of nonlinear elasticity and reaches a range of structural parameters in which the structure is inherently unstable. In this paper we identify and analyze the general properties of these paths by density functional theory. Special examples include vanadium, cobalt and copper, and the epitaxial path is used to identify an epitaxial film as related uniquely to a bulk phase.Comment: RevTeX, 4 pages, 4 figures, submitted to Phys. Rev. Let

Novel methods for in vitro modeling of pancreatic cancer reveal important aspects for successful primary cell culture

Background: Pancreatic cancer remains a fatal disease. Experimental systems are needed for personalized treatment strategies, drug testing and to further understand tumor biology. Cell cultures can serve as an excellent preclinical platform, but their generation remains challenging. Methods: Tumor cells from surgically removed pancreatic ductal adenocarcinoma (PDAC) specimens were cultured under novel protocols. Cellular growth and composition were analyzed and culture conditions were continuously optimized. Characterization of cell cultures and primary tumors was performed via hematoxylin and eosin (HE) and immunofluorescence (IF) staining. Results: Protocols for two- and three-dimensional PDAC primary cell cultures could successfully be established. Primary cell culture depended on dissociation techniques, growth factor supplementation and extracellular matrix components containing Matrigel being crucial for the transformation to three-dimensional PDAC organoids. The generated cultures showed to be highly resemblant to established PDAC primary cell cultures. HE and IF staining for cell culture and corresponding primary tumor characterization could successfully be performed. Conclusions: The work presented herein shows novel and effective methods to successfully establish primary PDAC cell cultures in a distinct time frame. Factors contributing to cell growth and differentiation could be identified with important implications for further primary cell culture protocols. The established protocols might serve as novel tools in personalized tumor therapy

Smoking, season, and detection of chlamydia pneumoniae DNA in clinically stable COPD patients

BACKGROUND: The prevalence and role of Chlamydia pneumoniae in chronic obstructive pulmonary disease (COPD) remain unclear. METHODS: Peripheral blood mononuclear cells were obtained from 100 outpatients with smoking-related, clinically stable COPD, and induced sputum was obtained in 62 patients. RESULTS: Patients had mean age (standard deviation) of 65.8 (10.7) years, mean forced expiratory volume in one second of 1.34 (0.61) L, and 61 (61.0%) were male. C. pneumoniae nucleic acids were detected by nested polymerase chain reaction in 27 (27.0%). Current smoking (odds ratio {OR} = 2.6, 95% confidence interval {CI}: 1.1, 6.6, P = 0.04), season (November to April) (OR = 3.6, 95% CI: 1.4, 9.2, P = 0.007), and chronic sputum production (OR = 6.4, 95% CI: 1.8, 23.2, P = 0.005) were associated with detection of C. pneumoniae DNA. CONCLUSIONS: Prospective studies are needed to examine the role of C. pneumoniae nucleic acid detection in COPD disease symptoms and progression

Exon expression arrays as a tool to identify new cancer genes

Background: Identification of genes that are causally implicated in oncogenesis is a major goal in cancer research. An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts. Here we report on a strategy to screen in a global fashion for such exon-skipping events using PAttern based Correlation (PAC). The PAC algorithm has been used previously to identify differentially expressed splice variants between two predefined subgroups. As genetic changes in cancer are sample specific, we tested the ability of PAC to identify aberrantly expressed exons in single samples. Principal Findings: As a proof-of-principle, we tested the PAC strategy on human cancer samples of which the complete coding sequence of eight cancer genes had been screened for mutations. PAC detected all seven exon-skipping mutants among 12 cancer cell lines. PAC also identified exon-skipping mutants in clinical cancer specimens although detection was compromised due to heterogeneous (wild-type) transcript expression. PAC reduced the number candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate. Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs). Conclusions: The ability of PAC to enrich for mutated transcripts and to identify known and novel genetic changes confirms its suitability as a strategy to identify candidate cancer genes

Alignment of the CMS tracker with LHC and cosmic ray data

© CERN 2014 for the benefit of the CMS collaboration, published under the terms of the Creative Commons Attribution 3.0 License by IOP Publishing Ltd and Sissa Medialab srl. Any further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation and DOI.The central component of the CMS detector is the largest silicon tracker ever built. The precise alignment of this complex device is a formidable challenge, and only achievable with a significant extension of the technologies routinely used for tracking detectors in the past. This article describes the full-scale alignment procedure as it is used during LHC operations. Among the specific features of the method are the simultaneous determination of up to 200 000 alignment parameters with tracks, the measurement of individual sensor curvature parameters, the control of systematic misalignment effects, and the implementation of the whole procedure in a multi-processor environment for high execution speed. Overall, the achieved statistical accuracy on the module alignment is found to be significantly better than 10μm