Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum

A cDNA of Trichoderma harzianum (chit42), coding for an endochitinase of 42 kDa, has been cloned using synthetic oligonucleotides corresponding to aminoacid sequences of the purified chitinase. The cDNA codes for a protein of 423 amino acids. Analysis of the N-terminal amino-acid sequence of the chitinase, and comparison with that deduced from the nucleotide sequence, revealed post-translational processing of a putative signal peptide of 22 amino acids and a second peptide of 12 amino acids. The chit42 sequence presents overall similarities with filamentous fungal and bacterial chitinases and to a lesser extent with yeast and plant chitinases. The deduced aminoacid sequence has putative catalytic, phosphorylation and glycosylation domains. Expression of chit42 mRNA is strongly induced by chitin and chitin-containing cell walls and is subjected to catabolite repression. Southern analysis shows that it is present as a single-copy gene in T. harzianum. chit42 is also detected in several tested mycoparasitic and non-mycoparasitic fungal strains.     

The infectivity of herpes simplex virus DNA in rat embryo cells is enhanced synergistically by DMSO and glucose

Transfection of HSV DNA into rat embryo cells (previously an inefficient procedure) is enhanced more than 30-fold by simultaneous treatment with 20% glucose and 10% DMSO 4 h after transfection. The action of these compounds was efficient only when used synergistically.     

Efficient in vivo transfection and safety profile of a CpG-free and codon optimized luciferase plasmid using a cationic lipophosphoramidate in a multiple intravenous administration procedure

As any drug, the success of gene therapy is largely dependent on the vehicle that has to selectively and efficiently deliver therapeutic nucleic acids into targeted cells with minimal side-effects. In the case of chronic diseases that require a life-long treatment, non-viral gene delivery vehicles are less likely to induce an immune response, thereby allowing for repeated administration. Beyond the gene delivery efficiency of a given vector, the nature of nucleic acid constructs also has a central importance in gene therapy protocols.Herein, we investigated the impact of two firefly luciferase encoding plasmids on the transgene expression profile following systemic delivery of lipoplexes in mice, as well as their potential to be safely and efficiently readministered. Whereas pTG11033 plasmid is driven by a strong ubiquitous cytomegalovirus promoter, pGM144 plasmid, which has been designed to avoid inflammation and provide sustained transgene expression in lungs, is CpG-free and is under control of the human elongation factor-1 alpha promoter.Combined to the efficient cationic lipophosphoramidate BSV4, bioluminescence data showed that both plasmids were mostly expressed in the lungs of mice following a primary injection of lipoplexes. However, mice transfected with pGM144 exhibited a higher and more sustained transgene expression than those treated with pTG11033. Repeated administration studies revealed that several injections of lipoplexes could lead to similar transgene expression profiles if an interval of several weeks between subsequent injections was respected. A transient hepatotoxicity and a partial inflammatory response were caused by lipoplex injection, irrespective of the plasmid used.Altogether, these results indicate that repeated systemic administration of lipophosphoramidate-based lipoplexes in mice conducts to an effective lung transfection without serious side effects, and highlight the need to use long-lasting expressing and well tolerated plasmids in order to efficiently renew transgene expression by the successive doses.     

Introduction of nonselectible 2μ plasmid into [cir°] cells of the yeast S. cerevisiae by DNA transformation and in vivo site-specific resolution

Summary The 2 DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2 DNA therefore cannot be achieved, and the intracellular presence of 2 can only be assessed by molecular analysis of the DNA complement. In addition, 2 alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo sites-pecific recombination between the endogenous 2 DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2 repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2 plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2 DNA plasmid. This system can be utilized to introduce 2 DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.