Stereospecificity in the biosynthesis of papaverine

The stereochemistry of hydrogen removal from C-3 and C-4 in the aromatization of the heterocyclic ring of papaverine has been determined by incorporation experiments with stereospecifically tritiated norreticulines.     

The Quiescent-Cell Expression System for Protein Synthesis in Escherichia coli

The quiescent-cell expression system is a radical alternative to conventional fermentation for protein overproduction in Escherichia coli. It is dependent on the controlled overexpression of a small RNA called Rcd in hns mutant strains to generate nongrowing, quiescent cells which are not nutrient limited. Quiescent cells no longer produce biomass and have their metabolic resources channelled toward the expression of plasmid-based genes. The biosynthetic capacity of the system is demonstrated by its ability to express chloramphenicol acetyltransferase to more than 40% of total cell protein. Quiescent cells may provide an ideal environment for the expression of toxic as well as benign proteins.     

Identification of the 12-O-tetradecanoylphorbol-13-acetate-responsive enhancer of the MS gene of the Epstein-Barr virus.

We previously located two 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive enhancers, MSTRE-I and MSTRE-II, in the upstream sequence of the MS gene of Epstein-Barr virus (Liu, Q., and Summers, W.C. (1989) J. Virol. 63, 5062-5068). The core sequence of the MSTRE-I enhancer is now determined to be between -718 and -708 of the upstream sequence of the MS gene. The activity of the enhancer is also sensitive to its immediate surrounding sequence on either side. A single copy of a 30-base pair (bp) fragment containing the MSTRE-I sequence was able to confer TPA responsiveness upon the MS promoter even in the absence of an AP-1 binding site. Multiple tandem copies of this 30-bp fragment, regardless of their relative orientations to each other, could function synergistically to enhance the MS promoter activity. At least two copies of the 30-bp fragment were required to bestow TPA induction upon the thymidine kinase gene promoter of herpes simplex virus type 1. The MSTRE-I sequence could also be bound by a Fos-GCN4 chimeric protein but with an affinity much lower than that between the chimeric protein and the AP-1 binding site. This MSTRE-I region has strong homology to one of the TPA-responsive elements (the ZII domain) in the upstream sequence of the EBV BZLF1 gene. In addition, a putative negative regulatory region or silencer was found immediately downstream of the MSTRE-I enhancer. This potential silencer region contains a 14-bp sequence that is homologous to the silencer consensus sequence of the BZLF1 gene. Therefore, the regulation of the MS gene may share the same pathway with the immediate early gene BZLF1.     

Comparative physical mapping of targeted regions of the rat genome

The comparative mapping and sequencing of vertebrate genomes is now a key priority for the Human Genome Project. In addition to finishing the human genome sequence and generating a `working draft' of the mouse genome sequence, significant attention is rapidly turning to the analysis of other model organisms, such as the laboratory rat (Rattus norvegicus). As a complement to genome-wide mapping and sequencing efforts, it is often important to generate detailed maps and sequence data for specific regions of interest. Using an adaptation of our previously described approach for constructing mouse comparative and physical maps, we have established a general strategy for targeted mapping of the rat genome. Specifically, we constructed a framework comparative map of human Chromosome (Chr) 7 and the orthologous regions of the rat genome, as well as two large (>1-Mb) P1-derived artificial chromosome (PAC)-based physical maps. Generation of these physical maps involved the use of mouse-derived probes that cross-hybridized with rat PAC clones. The first PAC map encompasses the cystic fibrosis transmembrane conductance regulator gene (Cftr), while the second map allows a three-species comparison of a genomic region containing intra- and inter-chromosomal evolutionary rearrangements. The studies reported here further demonstrate that cross-species hybridization between related animals, such as rat and mouse, can be readily used for the targeted construction of clone-based physical maps, thereby accelerating the analysis of biologically interesting regions of vertebrate genomes. Received: 5 December 2000 / Accepted: 27 February 2001     

Nucleoprotein architecture and ColE1 dimer resolution: a hypothesis

Dimers of plasmid ColE1 are converted to monomers by site-specific recombination, a process that requires 240 bp of DNA ( cer ) and four host-encoded proteins (XerC, XerD, ArgR and PepA). Here, we propose structures for nucleoprotein complexes involved in cer –Xer recombination based upon existing knowledge of the structures of component proteins and computational analyses of protein structure and DNA curvature. We propose that, in the nucleoprotein complex at a single cer site, a PepA hexamer acts as an adaptor, connecting the heterodimeric recombinase (XerCD) to an ArgR hexamer. This provides a protein core around which the cer site wraps, its exact path being defined by strong sequence-specific interactions with ArgR and XerCD, weak interactions with PepA and sequence-dependent flexibility of cer . The initial association of single-site complexes (pairing) is proposed to occur via an ArgR–PepA interaction. Pairing between sites in a plasmid dimer is stabilized by DNA supercoiling and is followed by a structural isomerization to form a recombination-proficient synaptic complex. We propose that paired structures formed between sites in trans are too short-lived to permit synaptic complex formation. There is thus an energetic barrier to inappropriate recombination reactions. Our proposals are consistent with a wide range of experimental observations.     

Role of glycosylation in the transport of recombinant glycoproteins through the secretory pathway of lepidopteran insect cells

Cell lines established from the Lepidopteran insect Spodoptera frugiperda (e.g., Sf9) are used routinely as hosts for the expression of foreign proteins by baculovirus vectors. Previously, we showed that human tissue plasminogen activator (t-PA) was expressed, N-glycosylated, and secreted by Sf9 cells infected with a recombinant baculovirus (Jarvis DL, Summers MD: Mol Cell Biol 9:214-223, 1989). We also showed that t-PA secretion was blocked by tunicamycin (TM), an inhibitor of N-glycosylation, but not by castanospermine (CS) or N-methyldeoxynojirimycin, inhibitors of the initial steps in N-linked oligosaccharide processing. This suggested that the addition, but not the processing, of N-linked oligosaccharides is required for the secretion of recombinant t-PA from baculovirus-infected Sf9 cells. In this study, we present a more generalized evaluation of the role of N-glycosylation in the transport of recombinant glycoproteins through the Sf9 cell secretory pathway. Several different secretory or membrane-bound glycoproteins were expressed in control, TM-treated, or CS-treated Sf9 cells, and their appearance in the medium or on the cell surface was measured. The results showed that TM blocked the transport of some, but not all, of these proteins, whereas CS did not block the transport of any. This suggests that N-glycosylation is sometimes required for the transport of recombinant glycoproteins through the Sf9 secretory pathway, while processing of the oligosaccharides is not. At least two other proteins, p80 and p31, consistently coimmunoprecipitated with the nonglycosylated precursors of recombinant glycoproteins expressed in TM-treated Sf9 cells. Neither was antigenically related to any of the recombinant proteins. Relatively larger amounts of p80 and p31 were coprecipitated when transport was completely blocked by TM compared to when transport was only reduced or was unaffected. These results suggest that p80 and p31 block the transport of some nonglycosylated glycoprotein precursors in TM-treated Sf9 cells by binding to them and producing transport-incompetent heterooligomeric complexes. If this speculation is correct, then p80 and p31 are functionally analogous to the mammalian immunoglobulin heavy chain binding/glucose-regulated 78 kilodalton protein (BiP/GRP78).