Phleomycins are glycopeptide antibiotics related to bleomycins nucleotide sequence of the Sh ble gene and its polylinker and tallysomycins which are active at low concentrations on both fragments. prokaryotic and eukaryotic cells (1). We have cloned a phleomycin-resistance gene (Sh ble) from the genomic DNA of REFERENCES Streptoalloteichus hindustanus (ATCC 31158), a tallysomycin 1 Berdy,J. (1980) Handbook of.Antibiotic Compounds. Vol.4, pp. 459-491, producer. The Sh ble gene encodes a small acidic protein (MW

Thymidylate kinase (dTMP kinase; EC 2.7.4.9) catalyzes the phosphorylation of dTMP to form dTDP in both de novo and salvage pathways of dTTP synthesis. The nucleotide sequence of the tmk gene encoding this essential Escherichia coli enzyme is the last one among all the E. coli nucleoside and nucleotide kinase genes which has not yet been reported. By subcloning the 24.0-min region where the tmk gene has been previously mapped from the phage 236 (E9G1) of the Kohara E. coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), we precisely located tmk between acpP and holB genes. Here we report the nucleotide sequence of tmk, including the end portion of an upstream open reading frame (ORF 340) of unknown function that may be cotranscribed with the pabC gene. The tmk gene was located clockwise of and just upstream of the holB gene. Our sequencing data allowed the filling in of the unsequenced gap between the acpP and holB genes within the 24-min region of the E. coli chromosome. Identification of this region as the E. coli tmk gene was confirmed by functional complementation of a yeast dTMP kinase temperature-sensitive mutant and by in vitro enzyme assay of the thymidylate kinase activity in cell extracts of E. coli by use of tmk-overproducing plasmids. The deduced amino acid sequence of the E. coli tmk gene showed significant similarity to the sequences of the thymidylate kinases of vertebrates, yeasts, and viruses as well as two uncharacterized proteins of bacteria belonging to Bacillus and Haemophilus species.With the exception of (deoxy)thymidylate kinase (dTMP kinase or TMK), which catalyzes the phosphorylation of dTMP to form dTDP in the dTTP synthesis pathway, all of the Escherichia coli enzymes of the metabolic pathways for the de novo synthesis of deoxynucleotide precursors of DNA have been well characterized genetically and biochemically (34). dATP, dCTP, and dGTP are derived from the corresponding deoxyribonucleoside diphosphates by phosphorylation catalyzed by the nonspecific nucleoside diphosphate kinase. Because thymine deoxyribonucleotides have no ribonucleotide counterpart, additional reactions are required for dTTP synthesis involving successively dCTP deaminase, dUTPase, thymidylate synthase, dTMP kinase, and nucleoside diphosphate kinase to convert dCTP to dTTP. Since conversion of dTDP to dTTP is catalyzed by the nonspecific nucleoside diphosphate kinase, the dTMP kinase is the last specific enzyme of both de novo and salvage pathways of dTTP synthesis. Because the overall control of DNA synthesis is regulated in part by the finely adjusted pool of dTTP, it would be crucial to explore the expression and the regulation of the E. coli dTMP kinase gene.For Saccharomyces cerevisiae, knowledge of the function of the dTMP kinase gene (cdc8) comes from the studies of a cdc8 temperature-sensitive cell cycle mutant. The transcription of the yeast dTMP kinase gene has been shown to be cell cycle regulated (peaking at the S phase) and coexpressed with DNA ligase and thymidylate sy...

Abstract

Transdifferentiation in vivo is an attractive option for autologous replacement of pancreatic b cells in patients with type 1 diabetes. It has been achieved by adenoviral delivery of genes for transcription factors in the liver and pancreas of hyperglycaemic mice. However, these viral approaches are not clinically applicable. We used the hydrodynamic approach to deliver genes Pdx1, Ngn3 (Neurog3) and MafA singly and in combination to livers of normoglycaemic rats. Five expression plasmids were evaluated. Livers were removed 1, 3, 7, 14 and 28 days after gene delivery and assayed by quantitative PCR, semi-quantitative PCR and immunohistology. Functional studies on hyperglycaemic rats were performed. The highest and most sustained expression was from a CpG-depleted plasmid (pCpG) and a plasmid with an in-frame scaffold/matrix attachment region ((pEPI(CMV)). When Pdx1, Ngn3 and MafA were delivered together to normoglycaemic rats with these plasmids, insulin mRNA was detected at all time points and was w50-fold higher with pCpG. Insulin mRNA content of livers at days 3 and 7 was equivalent to that of a pancreas, with scattered insulin-positive cells detected by immunohistology, but levels declined thereafter. Prohormone convertase 1/3 was elevated at days 3 and 7. In hyperglycaemic rats, fasting blood glucose was lower at days 1, 3 and 7 but not thereafter, and body weight was maintained to day 28. We conclude that hydrodynamic gene delivery of multiple transcription factors to rat liver can initiate transdifferentiation to pancreatic b cells, but the process is reversible and probably requires more sustained transcription factor expression.

Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.