Nucleotide sequence of the primary origin of bacteriophage T7 DNA replication: relationship to adjacent genes and regulatory elements.

The 682-base-pair nucleotide sequence between positions 14.45 and 16.15 on the bacteriophage T7 DNA molecule has been determined. We can identify not only the sequence of the primary origin of DNA replication but also the termination of gene 1, all of genes 1.1 and 1.2, the start of gene 1.3, and a number of regulatory sequences. The endpoints of four deletion mutations that extend into this region have been determined. These mutations are inferred to have arisen by recombination between short homologous sequences, three of which ar T7 RNA polymerase promoters. The base changes of four point mutations in gene 1.2 have been identified. The sequence essential for initiation at the primary origin is located between the left endpoints of the two deletions D2 and D303. Sequence analysis of these mutants assigns the primary origin to a 129-base-pair segment between positions 14.73 and 15.05. This intergenic segment is A+T-rich (75%) and contains a single T7 gene 4 protein recognition site; it is preceded by two tandem T7 RNA polymerase promoters. A model for initiation of T7 DNA replication is presented.     

A tagging-via-substrate technology for detection and proteomics of farnesylated proteins

A recently developed proteomics strategy, designated tagging-via-substrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and/or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.     

Function of adenovirus terminal protein in the initiation of DNA replication.

An early event in the initiation of adenovirus DNA replication is the formation of a covalent complex between the 87,000-dalton adenovirus terminal protein precursor and 5'- dCMP (pTP-dCMP complex). Nuclear extracts prepared from adenovirus-infected HeLa cells catalyzed complex formation in the presence of ATP, Mg2+, and adenovirus DNA-protein complex but were not active when Pronase-treated DNA was used as template. The activity has been partially purified by chromatography on denatured DNA-cellulose and used to examine whether the 55,000-dalton terminal protein on adenovirus DNA is required for pTP-dCMP complex formation. Results obtained with either DNA-protein complex or Pronase-treated DNA were identical to those obtained using crude nuclear extracts. However, after treatment with piperidine to remove residual peptides. Pronase-treated DNA supported complex formation with the partially purified activity but not with the crude extracts. In addition, when a plasmid containing an origin of adenovirus DNA replication was used as template, the pTP-dCMP complex was formed provided the plasmid was linearized in such a way that the origin was located at the end of the molecule. Neither linearized plasmid DNA with an internal origin nor supercoiled plasmid DNA supported complex formation. Furthermore, after heat denaturation, the linear plasmid DNA still supported complex formation, again provided that the origin was located at the end of the molecule. The partially purified protein fraction supported a limited amount of DNA chain elongation, which permitted exact positioning of the initiation site. These results suggest that enzymes responsible for complex formation recognize a DNA sequence at the origin and that the terminal protein on the template DNA plays a subordinate role.     

Development of mesoporous silica nanomaterials as a vehicle for anticancer drug delivery

The development of delivery vehicles that would carry therapeutic agents selectively to cancer cells has become an important focus in biomedical research. Nanoparticles have received much attention because the advances made in this field have resulted in multiple biocompatible materials. In particular, mesoporous silica nanoparticles (MSNs) offer a solid framework with porous structure and high surface area that allows for the attachment of different functional groups. In this article we discuss the different surface modifications made to MSNs that have allowed for the construction of targeted nanoparticles to enhance accumulation and uptake in target sites, the incorporation of nanomachines for controlled cargo release and the combination with superparamagnetic metals for MRI cell labeling. We also discuss biocompatibility, biodistribution and drug-delivery efficacy of MSNs. Finally, we mention the construction of multifunctional nanoparticles that combine all of the previously examined nanoparticle modifications.     

Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells

Rheb is a unique member of the Ras superfamily GTP-binding proteins. We as well as others previously have shown that Rheb is a critical component of the TSC/TOR signaling pathway. In fission yeast, Rheb is encoded by the rhb1 gene. Rhb1p is essential for growth and directly interacts with Tor2p. In this article, we report identification of 22 single amino acid changes in the Tor2 protein that enable growth in the absence of Rhb1p. These mutants also exhibit decreased mating efficiency. Interestingly, the mutations are located in the C-terminal half of the Tor2 protein, clustering mainly within the FAT and kinase domains. We noted some differences in the effect of a mutation in the FAT domain (L1310P) and in the kinase domain (E2221K) on growth and mating. Although the Tor2p mutations bypass Rhb1p's requirement for growth, they are incapable of suppressing Rhb1p's requirement for resistance to stress and toxic amino acids, pointing to multiple functions of Rhb1p. In mammalian systems, we find that mammalian target of rapamycin (mTOR) carrying analogous mutations (L1460P or E2419K), although sensitive to rapamycin, exhibits constitutive activation even when the cells are starved for nutrients. These mutations do not show significant difference in their ability to form complexes with Raptor, Rictor, or mLST8. Furthermore, we present evidence that mutant mTOR can complex with wild-type mTOR and that this heterodimer is active in nutrient-starved cells.