Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

We have isolated a nucleus-basal body complex from Chlamydomonas reinhardtii.The complex is strongly immunoreactive to an antibody generated against a major protein constituent of isolated Tetraselmis striata flagellar roots (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, J. Cell Biol.,. Electrophoretic and immunoelectrophoretic analysis indicates that, like the Tetraselmis protein, the Chlamydomonas antigen consists of two acidic isoforms of ~20 kD. Indirect immunofluorescent staining of nucleus-basal body complexes reveals two major fibers in the connector region, one between each basal body and the nucleus. The nucleus is also strongly immunoreactive, with staining radiating around much of the nucleus from a region of greatest concentration at the connector pole. Calcium treatment causes shortening of the connector fibers and also movement of nuclear DNA towards the connector pole. Electron microscopic observation of negatively stained nucleus-basal body complexes reveals a cluster of ~6-nm filaments, suspected to represent the connector, between the basal bodies and nuclei.A mutant with a variable number of flagella, vfl-2-220, is defective with respect to the nucleus-basal body association. This observation encourages us to speculate that the nucleusbasal body union is important for accurate basal body localization within the cell and/or for accurate segregation of parental and daughter basal bodies at cell division.A physical association between nuclei and basal bodies or centrioles has been observed in a variety of algal, protozoan, and metazoan cells, although the nature of the association, in terms of both structure and function, has been obscure. We believe it likely that fibrous connectors homologous to those described here for Chlamydomonas are general features of centriole-bearing eucaryotic cells.The basal body cycle of Chlamydomonas resembles the classical centriole cycle in most, if not all, essential respects, and so an analysis of the Chlamydomonas basal body cycle may provide information about centriole cycles in general. The cytological behavior of centrioles during the cell cycle is well known (43). Landmark events in the centriole cycle include duplication during interphase, migration to the nuclear poles before mitosis, presence at the mitotic spindle poles, and equal segregation to daughter cells at division. Despite a very large body of literature on centriole structure and cytology, the cellular function of centrioles remains obscure, and most of the questions raised when centrioles were observed more than one hundred years ago remain unanswered. Why are centrioles maintained in such a wide variety of organisms? How do centriole duplication and segregation occur? Is proper subcellular localization of centrioles important to their function?Protozoan centrioles are generally referred to as basal bodies, and we will follow this convention throughout this paper. But the reader should bear in mind that in many cell types, both metazoan and protozoan, a basal body or centri...

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the “valley of death” by bridging basic to clinical sciences.

Epitope tagging is a recombinant DNA method by which a protein encoded by a cloned gene is made immunoreactive to a known antibody. This review discusses the major advantages and limitations of epitope tagging and describes a number of recent applications. Major areas of application include monitoring protein expression, localizing proteins at the cellular and subcellular levels, and protein purification, as well as the analysis of protein topology, dynamics and interactions. Recently the method has also found use in transgenic and gene therapy studies and in the emerging fields of functional genomics and proteomics.

Abstract. Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrincontaining fibrous structures associated with the Chlamydomonas centrosome.

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S rc is a membrane-bound tyrosine kinase(1) that has been implicated in the progression of many human cancers (2,3) including tumors of the breast, (4-6) kidney, (7) and prostate. (8) The Src kinase phosphorylates Cas (Crk-associated substrate) and activates mitogen-activated protein kinase (MAPK) to promote anchorage-independent growth and migration. (9)(10)(11)(12)(13)(14)(15)(16) These fundamental hallmarks of tumor-cell growth are required for metastasis and distinguish most cancer cells from their non-transformed precursors. (17,18) We have recently found that Src utilizes Cas to inhibit Fhl1 (four and a half LIM domains 1) expression, in order to promote non-anchored cell growth and migration. (19) Transfection studies show that Fhl1 specifically blocks non-anchored tumor-cell growth and migration, but does not affect 'normal' anchored cell growth. Therefore, Fhl1 acts as a true tumor suppressor rather than as a general mitotic inhibitor. (19) As the name indicates, Fhl1 consists of 'four and a half LIM domains'. Fhl1 can move between intercellular junctions, (20) focal adhesions, and the nucleus (21) to affect gene expression. (22,23) For example, Fhl1 associates with the recombination signal binding protein for immunoglobulin kappa J region (RBP-J)-DNA binding protein to modulate gene transcription. (22,23)