DNA cross-linking reagents are frequently unusually cytotoxic, and many, including the nitrogen mustards, are potent chemotherapeutic agents, presumably because DNA cross-links effectively block DNA replication. Most of these reagents form both interand intrastrand DNA cross-links, but it is unknown which is more effective at blocking repli cation and why. To evaluate the role of interstrand cr...

DNA cross-linking reagents are frequently unusually cytotoxic, and many, including the nitrogen mustards, are potent chemotherapeutic agents, presumably because DNA cross-links effectively block DNA replication. Most of these reagents form both inter- and intrastrand DNA cross-links, but it is unknown which is more effective at blocking replication and why. To evaluate the role of interstrand c...

Increasing evidence suggests that glutamate activates the generation of lactate from glucose in astrocytes; this lactate is shuttled to neurons that use it as a preferential energy source. We explore this multicellular "lactate shuttle" with a novel dual-cell, dual-gene therapy approach and determine the neuroprotective potential of enhancing this shuttle. Viral vector-driven overexpression of ...

We develop a murine retrovirus shuttle vector system for the efficient introduction of selectable and nonselectable DNA sequences into mammalian cells and recovery of the inserted sequences as molecular clones. Three protocols allow rapid recovery of vector DNA sequences from mammalian cells. Two of the methods rely on SV40 T-antigen-mediated replication of the vector sequences and yield thousa...

A human metallothionein (MT) gene was inserted into a bovine papillomavirus (BPV) vector. The chimeric vector (pMTII-BPV) transforms rodent fibroblasts to a cadmium-resistant phenotype. The resistance is due to the high level of expression of human MT-II in those cells. The vector is maintained in the cells as a free replicating plasmid, present at about 10--15 copies per cell. Transcription of...

The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartite geminivirus, was studied in cultured maize endosperm cells, and in the Black Mexican Sweet (BMS) maize cell line. Using in vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,0...

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a delta-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vect...

We constructed a pair of Nocardia-Escherichia coli shuttle vectors, pNV18 and pNV19, by combining the mycobacterial plasmid pAL5000 with the E. coli vector pK18 or pK19. These vectors have a number of useful features, including small size (4.4 kb), a multiple cloning site, and blue/white selection. To our knowledge, pNV18 and pNV19 are the first cloning vectors for practical use in Nocardia spp.

To show, as a model system, that mycobacteria can express heterologous luciferase genes and that bioluminescence can be a rapid method of measuring antimycobacterial activity, a bioluminescent form of Mycobacterium smegmatis was made by transformation with a Mycobacterium-Escherichia coli shuttle vector containing the luxAB genes from Vibrio harveyi. The antimycobacterial effects of antibiotics...