Construction Of Cassettes Research Articles

The elements of the locus of enterocyte effacement in human and wild mammal isolates of Escherichia coli: evolution by assemblage or disruption?

Escherichia coli is an excellent model for studying the evolution of pathogenicity since within one species various genes can be found in pathogenic islands and plasmids causing a wide spectrum of virulence. A collection of 122 strains from different human and wild mammal hosts were analysed by PCR and Southern hybridization for the presence of a subset of the genes included in the LEE (locus of enterocyte effacement). In the PCR analysis, two markers (cesT/eae and espB genes) were found together in more strains (25.4%) than either were found alone. The cesT/eae gene was less frequently found alone (8.2%) than was the espB gene (15.6%). Four regions of the LEE were analysed in a subsample of 25 strains using Southern hybridization. The four regions were all present (44%), all absent (12%) or present in different combinations (44%) in a given strain. The flanking regions of the LEE showed the highest rate of hybridization (in 72% of the strains). The results indicate that the LEE is a dynamic genetic entity, both the complete gene cluster and the individual genes. The genes that comprise this locus seem to be horizontally acquired (or lost) in an independent way and may control other functions in non-pathogenic E. coli lineages. In this way, horizontal transfer may allow the gradual stepwise construction of gene cassettes facilitating coordinate regulation and expression of novel functions.

Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe.

We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.

Construction, biological activity, and immunogenicity of synthetic envelope DNA vaccines based on a primary, CCR5-tropic, early HIV type 1 isolate (BX08) with human codons.

So far codon-optimized HIV-1 envelope genes have been investigated for the T cell line-adapted strain MN, which differs in several aspects from primary isolates. Envelopes of primary isolates may be more relevant for vaccine purposes. This article describes for the first time the engineering and characterization of four "humanized" genes encoding the secreted gp120/gp140, or the membrane-bound gp150/gp160, of a primary CCR5 tropic, clade B, clinical isolate HIV-1(BX08). The genes were built in fragments for easy cassette exchange of regions important for immunogenicity, function, and expression. The transcription and expression of the synthetic genes in mammalian cell lines were Rev independent and highly increased. Increased expression of membrane-bound gp160 induced a high cytopathic effect in U87.CD4.CCR5 cells. Gene gun and intramuscular DNA vaccination in mice induced a strong specific cytotoxic T lymphocyte response independent of the gene construct, expression level, or DNA immunization route. In contrast, the highest anti-gp120 antibody levels were induced by synthetic genes encoding the secreted glycoproteins followed by gp160/gp150. Unlike HIV-1(MN), HIV-1(BX08) V3 was not immune dominant. Despite the high antibody response only low and inconsistent neutralizing titers to the homologous HIV-1 isolate were measured. However, neutralization of SHIV89.6P could be obtained. Thus, the neutralizing epitopes on the cell line-adapted SHIV89.6P and HIV-1(MN) may be more antigenically available for the cross-neutralizing antibodies induced. In conclusion, complete "humanization" of the DNA vaccine genes failed to induce a consistent neutralizing antibody response, albeit expression and immunogenicity of the primary HIV-1 glycoproteins were greatly improved. Optimization in terms of improving neutralization may require further modifications of the DNA vaccine gene. The synthetic cassette construct described is a convenient tool developed to investigate this further.

Recent developments in the hammerhead ribozyme field

Developments in the hammerhead ribozyme field during the last two years are reviewed here. New results on the specificity of this ribozyme, the mechanism of its action and on the question of metal ion involvement in the cleavage reaction are discussed. To demonstrate the potential of ribozyme technology examples of the application of this ribozyme for the inhibition of gene expression in cell culture, in animals, as well as in plant models are presented. Particular emphasis is given to critical steps in the approach, including RNA site selection, delivery, vector development and cassette construction.

Initiation codon scanthrough versus termination codon readthrough demonstrates strong potential for major histocompatibility complex class I-restricted cryptic epitope expression.

Accumulating evidence shows that the repertoire of major histocompatibility complex class I-restricted epitopes extends beyond conventional translation reading frames. Previously, we reported that scanthrough translation, where the initiating AUG of a primary open reading frame is bypassed, is most likely to account for the presentation of cryptic epitopes from alternative reading frames within the influenza A PR/8/34 nucleoprotein gene. Here, we confirm and extend these findings using an epitope cassette construct that features two well-defined CD8(+) T cell (TCD8+) epitopes in alternative reading frames, each preceded by a single start codon. Expression of one epitope depends on scanning of the ribosome over the first AUG with translation initiation occurring at the second AUG. We find that scanthrough translation has great potency in our system, with its impact being modulated, as predicted, by the base composition surrounding the first initiation codon, the number of start codons preceding the point of alternate reading frame initiation, and the efficiency with which the epitope itself is generated. Additionally, we investigated the efficiency of eukaryotic translation termination codons, to assess codon readthrough as a mechanism for cryptic epitope expression from 3' untranslated regions. In contrast with initiation codons, eukaryotic stop codons appear to be highly efficient at preventing expression of epitopes encoded in 3' untranslated regions, suggesting that 3' untranslated regions are not a common source of cryptic epitope substrate. We conclude that scanthrough is a powerful mechanism for the expression of epitopes encoded in upstream alternative open reading frames that may contribute significantly to TCD8+ responses and to tolerance induction.

The CCAAT-binding Proteins CP1 and NF-I Cooperate with ATF-2 in the Transcription of the Fibronectin Gene

We have previously proposed a molecular interaction between the liver factors that bind to the cyclic AMP response element (CRE) and CCAAT sites of the fibronectin (FN) gene based on the following evidence: (i) the close spacing of 20 base pairs between CRE and CCAAT elements is conserved in the FN genes from rats, mice, and humans; (ii) footprinting competitions showed that CRE oligonucleotides are able to detach both liver factors; (iii) CCAAT binding and transcriptional activity of liver extracts are reduced when the distance between the CRE and CCAAT elements is increased; and (iv) CCAAT-binding is stimulated by the addition of a liver extract fraction containing the CRE-binding factor ATF-2. This report provides binding and immunochemical evidence that nuclear factor I (CTF/NF-I) and CP1 (NF-Y or CBF) are the only liver factors that bind to the -150 CCAAT element of the FN gene, forming distinct complexes. We show that these factors bind less efficiently to the CCAAT site of a FN promoter in which the -170 CRE has been disrupted by site-directed mutagenesis and that each element contributes positively to the liver transcriptional activity assessed in vitro with a G-less cassette construct and in vivo by transfection of hepatoma cells with CAT constructs. Furthermore, using a method that combines UV cross-linking and immunoprecipitation, we show that antibodies specific to ATF-2 are able to specifically precipitate protein-protein-DNA complexes containing NF-I and CP1. This simple method preserves weak macromolecular interactions, avoiding the disruptive electrophoresis conditions of gel mobility shifts assays.

Expression of small synthetic genes coding for hEGF, human epidermal growth factor, and CPTI II, serine proteinase inhibitor from Cucurbitacea, cloned in a novel expression/secretion vector in Saccharomyces cerevisiae.

Efficient synthesis of two small eukaryotic polypeptides of human and plant origin was carried out using a novel expression/secretion yeast vector, pYET. The yield was optimized in respect of the yeast strain, expression cassette construction, promoter regulation and culture conditions. Both cloned genes code for biotechnologically important proteins: human epidermal growth factor and a serine proteinase inhibitor from Cucurbitacea.

Leucine Zipper Dimerized Bivalent and Bispecific scFv Antibodies from a Semi-synthetic Antibody Phage Display Library

This report describes the construction of leucine zipper-based dimerization cassettes for the conversion of recombinant monomeric scFv antibody fragments to bivalent and bispecific dimers. A truncated murine IgG3 hinge region and a Fos or Jun leucine zipper were cloned into four scFv fragments previously isolated from a synthetic antibody phage display library. Cysteine residues flanking the zipper region were introduced to covalently link dimerized scFv fragments. The secreted fusion proteins were shown to spontaneously and efficiently form stable Fos-Fos or Jun-Jun homodimers in the Escherichia coli periplasm at levels comparable to their monovalent counterparts. The bivalent (scFv)2 fragments performed well in enzyme-linked immunosorbent assay, flowcytometric, and immunohistochemical analysis. Fos and Jun homodimer (scFv)2 antibodies with different specificities could be reduced, reshuffled, and reoxidized to form preparations of functional bispecific (scFv)2 Fos-Jun heterodimers. These Fos and Jun fusion protein cassettes provide a universal basis for the construction of dimeric scFv antibodies with enhanced avidity or dual specificity.

Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis

Several antibiotic-resistance gene cassettes and Ω elements for Escherichia coli vector construction include the aacC1, aadA +, bla, cat, nptII and tet gene cassettes, and also the Ω-Gm, Ω-Sm, Ω-Ap, Ω-Cm, Ω-Km and Ω-Tc elements. Both cassettes and elements are flanked by pBluescriptII plasmid multiple cloning sites (MCS) duplicated in inverted (symmetric MCS) or direct (tandem MCS) orientation. Genes that were modified in order to remove sites for the most common restriction endonucleases from their coding regions (except aacC1 and aadA + ) were used for cassette and Ω-element construction.

Multiple Cis-Acting DNA Regulatory Elements Mediate Hepatic Angiotensinogen Gene Expression

Angiotensinogen is the glycoprotein precursor of angiotensin II, an octapeptide hormone important for the regulation of blood pressure and volume homeostasis. The gene encoding angiotensinogen is expressed in liver and several other tissues, providing a model gene for understanding the role of cis-acting DNA control elements and trans-acting factors in tissue-type specific gene expression. To identify DNA control elements in the rat angiotensinogen gene we prepared an array of fusion genes consisting of either 5' or 3'-deleted sequences of the 5'-flanking region of the gene linked to a firefly luciferase reporter gene and analyzed the relative cellular specificity of expression of these genes after their introduction into hepato-carcinoma cells (Hep G2) that do express and placental cells (JEG-3) that do not express the endogenous angiotensinogen gene. Six transcriptionally active elements were found within 688 base pairs of 5'-flanking DNA. The interactions of DNA binding proteins with these elements was demonstrated by their specific protection to digestion with DNase I in the presence of liver cell extracts. The orientation and spatial requirements for transcription of two of the elements were analyzed further by the construction and expression of synthetic oligonucleotide cassettes incorporating the sequences of these elements when linked to a homologous (angiotensinogen) or a heterologous Simian virus 40 promoter and enhancer. One element located between 60 and 108 base pairs from the start of gene transcription functioned either as a silencer or an enhancer of transcription (SOAP box element), depending upon the distance from the angiotensinogen and viral gene promoters. Moreover, the SOAP box element demonstrated enhancer activity in JEG-3 cells when linked to the Simian virus 40 early promoter. An oligonucleotide mutation of the SOAP box element interfered with protein binding in a gel mobility shift assay and this mutant was transcriptionally inactive in both homologous and heterologous promoters. These observations indicate that expression of the angiotensinogen gene in liver cells is coordinately regulated by multiple cis-acting elements that interact with DNA binding proteins.

13] Simultaneous synthesis and biological applications of DNA fragments: An efficient and complete methodology

Publisher Summary This chapter discusses the simultaneous synthesis and biological applications of deoxyribo nucleic acid (DNA) fragment that is an efficient and complete methodology. One must first analyze the specific situation and consider factors of confidence, speed, flexibility, budget, available know-how, and annual demand. Suppose the decision was made to synthesize oligonucleotides in-house and to use them for the construction of genes or mutagenesis cassettes, or just for a variety of smaller projects. The basic idea of this approach is rather simple. Instead of using small beads or fibers, as in the classical approaches to peptide and nucleic acid synthesis, this chapter employs “segmental supports,” non interchangeable entities, as the immobilizing support. This type of support allows one to elongate simultaneously those oligonucleotide chains that require the same nucleotide. This chapter concludes with the discussion on the construction of DNA double strands. DNA double strands constructed as described basically represent pieces of DNA built up from small cassettes. New sequence variants are easily made, in high yields, by exchange, insertion, or deletion of two or more fragments. Most of the fragments can be reused many times.

Effect of vertical cassette holder design and construction on scatter-to-primary radiation ratios.

This paper analyzes the contribution to scattered radiation at the film plane by individual components of a conventional wall-mounted cassette holder. With grid technique, scatter from the front panel of the cassette holder is effectively reduced, but considerable scatter reaches the film plane from the automatic exposure control pickup interposed between the grid and the film cassette. With air-gap technique (grid removed), the front panel of the cassette holder and the automatic exposure control pickup contribute equally to increased scattered radiation at the film plane. The contribution to the total scatter from the air-gap positioning plate is less significant.

Thermal conditions of electronic instruments of cassette construction with forced ventilation

A general approach is considered for the calculation of the temperature field of a ventilated electronic instrument of cassette construction through the reduction of its heated zone to a uniform body. An approximate solution of the problem is proposed and the results of calculations are compared with experimental data.