Peter T. Lomedico

Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli

We have constructed a set of expression vectors which contain synthetic DNA sequences comprising a computer-generated model ribosomal binding site located downstream from the tightly regulated phage lambda pL. promoter. These vectors have been used in several laboratories to produce significant amounts of eukaryotic and prokaryotic gene products in Escherichia coli, either as fusion proteins (with two to nine extra N-terminal amino acids) or as proteins containing the naturally occurring amino terminus. For inserting DNA sequences downstream of an initiation codon, we used synthetic oligonucleotides to introduce multiple-use restriction sites recognized by EcoRI, BamHI and ClaI which generate termini complementary to those of a variety of enzymes (e.g., EcoRI, MboI, TaqI, and HpaII), in addition to their own. A set of three of these vectors was made to accommodate all three translational reading frames. In combination, the features of these vectors afford useful advantages over expression vectors previously described, especially for the application of shot-gun cloning of genomic DNA to generate expression libraries.

Cloning and nucleotide sequence of newcastle disease virus hemagglutinin-neuraminidase mRNA: Identification of a putative sialic acid binding site

The avian pathogen Newcastle disease virus (NDV) is a member of the Paramyxoviridae, a family which includes Sendai virus, simian virus 5 (SV5) and the human virus parainfluenza 3 (para-3). These enveloped viruses contain a single-stranded RNA genome of negative sense, 15-18 kb in length, encased in a helical nucleocapsid. Six major messenger RNA species are transcribed sequentially by the virion-associated RNA polymerase. They are capped, methylated, and polydenylated by virion proteins (Huang et a/., 1971; Colonno and Stone, 1976). Organization of the NDV genome has been investigated indirectly by both uv-inactivation mapping and by the analysis of polycistronic transcripts (Collins et a/., 1980; Wilde and Morrison, 1984). Both studies indicate that the gene order is identical to that of Sendai virus, which has been determined to be 3’-NP-P-M-F-HN-L-5’ by nucleotide sequence analysis of genomic clones (Blumberg et a/., 1984, 1985a,b). NP and the phosphoprotein P are components of the viral nucleocapsid, L is an RNAdependent RNA polymerase, and M is a protein of undetermined function associated with the inner surface of the viral membrane (reviewed in Kingsbury, 1985). Paramyxovirus glycoproteins are functionally similar to the well-studied surface glycoproteins of influenza (Ward, 1981; Colman and Ward, 1985). They possess cell fusion, hemagglutination and neuraminidase activities (Hsu et a/., 1979; Scheid and Choppin, 1974; Merz et a/., 1981), and constitute the spikes which protrude from the viral envelope (Choppin and Compans, 1975). The F glycoprotein, which is responsible for the paramyxovirus’ ability to fuse with the host cell membrane, is activated by a proteolytic cleavage event (Scheid and

Eukaryotic ribosomes can recognize preproinsulin initiation codons irrespective of their position relative to the 5' end of mRNA.

The functional assay of a eukaryotic mRNA, into which additional AUG codons have been introduced by in vitro mutagenesis, shows that a translational initiation site need not necessarily be the nearest AUG codon to the 5′ end of a mRNA. Hence, the sequence surrounding the AUG, and not simply its position relative to the 5′ end of mRNA, appears to be important in determining initiation efficiency.

Chapter 18. Interleukin 1

Publisher Summary This chapter discusses the biochemical characterization and the physiological role of interleukin 1 (IL-1). IL-1 is a molecule that possesses a wide variety of biological activities. Biochemical studies reveal that IL-1 produced by stimulated murine macrophages and macrophage tumor cell lines is a single polypeptide chain. Human IL-1 has been shown to be produced by stimulated normal human peripheral blood leukocytes or monocytes, leukemic cells, and a number of human tumor cell lines. Murine and human keratinocytes have been shown to produce an IL-1-like activity designated epidermal T-cell activating factor. This factor exhibits some of the activities of IL-1. IL-1 appears to play a key role in the activation of T-lymphocytes. The processes triggered by IL-1 may include both the secretion of IL-2 and induction of receptors for this growth factor. IL-1 can also modulate B-cell function. However, the nature of the effect can vary depending on the presence of other lymphocyte-derived signals and the type of B-cell stimulus. An effect of IL-1 on fibrinogen and haptoglobin synthesis has also been discussed in the chapter. IL-1 may also be a modulator of the fibroblast proliferation. IL 1 has also significant effects on the cartilage, bone, and synovial membrane. IL-1 is thought to be an important mediator of immune cell interactions and to contribute to a number of disease manifestations and physiological processes. Thus, agents that modulate either IL-1 production or IL-1 activity on the target tissues may be therapeutically beneficial and have been the focus of a number of investigations. This information and additional developments in IL-1 research should lead to an understanding of the mechanism, by which IL-1 induces each of its effector activities, of the role of IL-1 in various disease states and of the means, by which specific activities of IL-1 can be modulated.