A Simple Solid-Phase Based Purification Procedure for Oligodeoxynucleotides

Abstract

A two-cartridge method for routine purification of DNA oligomers has been investigated. The full-length target oligonucleotides are purified using a method that select for intact 3′- and 5′-termini. The procedure results in purified DNA without the use of PAGE gels or HPLC.     

Solid Supported Chemical 5′-Phosphorylation of Oligodeoxy-Ribonucleotides that can be Monitored by Trityl Cation Releasi Application to Gene Synthesis

Abstract

The gene for human lysozyme was assembled from oligonucleotides that were chemically phosphorylated with a novel phosphorylation reagent.     

Phagocytic ability declines with age in adult Drosophila hemocytes

Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age‐related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1‐week‐ and 5‐week‐old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.     

Histone deacetylases play distinct roles in telomeric VSG expression site silencing in African trypanosomes

African trypanosomes evade the host immune response through antigenic variation, which is achieved by periodically expressing different variant surface glycoproteins (VSGs). VSG expression is monoallelic such that only one of approximately 15 telomeric VSG expression sites (ESs) is transcribed at a time. Epigenetic regulation is involved in VSG control but our understanding of the mechanisms involved remains incomplete. Histone deacetylases are potential drug targets for diseases caused by protozoan parasites. Here, using recombinant expression we show that the essential Trypanosoma brucei deacetylases, DAC1 (class I) and DAC3 (class II) display histone deacetylase activity. Both DAC1 and DAC3 are nuclear proteins in the bloodstream stage parasite, while only DAC3 remains concentrated in the nucleus in insect‐stage cells. Consistent with developmentally regulated localization, DAC1 antagonizes SIR2rp1‐dependent telomeric silencing only in the bloodstream form, indicating a conserved role in the control of silent chromatin domains. In contrast, DAC3 is specifically required for silencing at VSG ES promoters in both bloodstream and insect‐stage cells. We conclude that DAC1 and DAC3 play distinct roles in subtelomeric gene silencing and that DAC3 represents the first readily druggable target linked to VSG ES control in the African trypanosome.     

Seed glucosinolates of fourteen wild Brassica species

Seed glucosinolates have been determined for 14 wild Brassica species, by micro-scale GC analysis of silylated derivatives. Of these, 12 were investigated for the first time. The majority of taxa exhibited high alkenylglucosinolate levels, although prop-2-enylglucosinolate appears to be generally absent. Other known methionine-derived glucosinolates predominate in B. tournefortii, B. elongata and B. deflexa. Phenylalanine-derived 4-hydroxybenzylglucosinolate is characteristic of section Brassicaria plants and represents the first finding of this glucosinolate in authenticated Brassica material.     

A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara(MVA)

Vectored vaccines based on highly attenuated modified vaccinia Ankara(MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a nonplaque-purified preparation of MVA in a continuous anatine suspension cell line(CR.pIX) in chemically defined medium.The novel isolate(MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVACR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA:although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.