Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications.

The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. We have identified a bicoid-related homeobox gene, Ptx3, a member of the Ptx-subfamily, that is uniquely expressed in these neurons. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of Ptx3-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxy-dopamine-lesioned rats, an animal model for this disease. Thus, Ptx3 is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage.

https://www.pnas.org/content/pnas/94/24/13305.full.pdf

A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons

Publisher Summary This chapter discusses the types, effects, and properties of interferons (IFN). Interferons are classified into two types, I and II, on the basis of distinct biological, biochemical, and serological properties. Type II IFN, which is acid-labile, is secreted by T lymphocytes after immune or mitogenic stimulation. Analyses at the genomic level have revealed only one gene for type II IFN, namely—IFN-γ, which shows no clear relationship to the genes of type I IFN and is located on a different chromosome than the latter. Type I IFNs are induced by viral infection or the treatment of a variety of cells with poly(I)·poly(C) and are usually acid-stable. The genes encoding type I IFNs are the members of a superfamily. All are located on one chromosome and comprise the IFN-β and IFN-α families, which diverged 200-300 million years ago or more. In ungulates, the IFN-β family comprises several genes and in man and mouse, only one. The recently described IFN-β 2 , while cross-reacting with IFN-β antibodies, is apparently not related to the IFN-β at the genetic level. The IFN-α family comprises at least two subfamilies, IFN-α I and IFN-α II , which diverged 120 million years ago or more, clearly before the mammalian radiation.

The interferon genes.

Plasmid vector pBR322 and its special-purpose derivatives--a review.

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are approximately 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5-7 days) and suitable for synthesizing long segments of DNA (5-6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.

/pdf/a-simple-rapid-high-fidelity-and-cost-effective-pcr-based-1f7jo6zbpv.pdf

A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences

A 511-base pair DNA fragment encoding human interferon-alpha 2 has been chemically synthesised and expressed from a lac UV5 and a synthetic trp promoter in Escherichia coli. The synthesis involved preparation of 68 oligodeoxyribonucleotides and their enzymic ligation. The product expressed from the trp promoter system had high antiviral activity and displayed biological effects similar to those of Namalwa interferon on natural killer cell activity and in a Daudi cell growth inhibition assay. E.coli minicells containing plasmid DNA with the synthetic IFN-alpha 2 gene under trp promoter control produce a protein with the same electrophoretic mobility as a sample of authentic IFN-alpha 2. The protein from E.coli cross-reacts with the monoclonal antibody NK-2 and was readily purified, close to homogeneity, by immunoadsorption chromatography on NK-2 sepharose.

/pdf/chemical-synthesis-of-a-human-interferon-a2-gene-and-its-133sfx4fya.pdf

Chemical synthesis of a human interferon-α2, gene and its expression in Escherichia coli

An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system.

The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.

Functionally important conserved amino-acids in interferon-α2 identified with analogues produced from synthetic genes

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Chemical synthesis of a human interferon-α2, gene and its expression in Escherichia coli

The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.

Functionally important conserved amino-acids in interferon-α2 identified with analogues produced from synthetic genes