The Escherichia coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. The regulation of the nrd operon has been observed to be very complex. The specific binding of two proteins to the nrd regulatory region and expression of mutant nrd-lac fusions that do not bind these proteins are described. A partially purified protein from an E. coli cell extract was previously shown to bind to the promoter region and to regulate transcription of the nrd operon (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990 starved cells. The protein extract from a thymine-starved culture protected several sites in the promoter region from DNase I digestion (47). Here, we report the identity of this protein as the E. coli factor for inversion stimulation (Fis).Fis is an 11.2-kDa protein initially identified by its ability to stimulate Hin-, Gin-, and Cin-mediated DNA inversion (18,23,25). The involvement of Fis in other site-specific recombinations, including excision and integration recombination reactions of bacteriophage X DNA, was also demonstrated (3,4,44). Fis has also been shown to be a transcriptional activator at rRNA and tRNA promoters in E. coli (6,38). In vitro footprinting experiments show protection of specific sites in the E. coli oriC sequence by Fis (17), and examination of DNA synthesis in fis gyrB double mutants indicates a synergistic involvement of Fis and DNA gyrase in the initiation of DNA replication at oriC (14).The fis gene has been cloned, sequenced, and mapped to 72 min on the E. coli chromosome (23,26). The gene encodes an 11,239-Da, 98-amino-acid polypeptide. The fis operon promoter has been located 1 kb upstream of the fis start codon (36), and Fis synthesis is autoregulated. Transcription of fis is controlled as part of the stringent response. A degenerate consensus binding site for Fis has been proposed: (G/T) nnYRnn(A/T)nnYRnn(C/A) (where Y is a pyrimidine, R is a purine, and n is any nucleotide) (22). However, local DNA topology and the ability of the DNA duplex to bend, accommodating the helix-turn-helix DNA-binding domains of the Fis dimer, appear to be more important in determining Fis binding at any given site (51) than the sequence per se. In activating transcription of stable RNA operons in E. coli, binding of Fis to upstream activating sequences lowers the dissociation constant of the RNA polymerase-promoter complex (34).

We have isolated mouse , opioid receptor genomic clones (termed MOR) containing the entire amino acid coding sequence correspondin to rat MOR-1 cDNA, including additional 5' flanking sequence. The mouse MOR gene is >53 kb long, and the coding sequence is divided by three introns, with exon junctions in codons 95 and 213 and between codons 386 and 387. The first intron is >26 kb, the second is 0.8 kb, and the third is >12 kb.

Spot 14 (S14) is a protein whose mRNA is rapidly up-regulated by lipogenic stimuli including thyroid hormone and a high-carbohydrate diet. Previous investigation into the role of S14 suggested that it is involved in de novo lipogenesis. Knockout of the gene in mice has given further support to this hypothesis. The lack of S14 in different tissues resulted in varying phenotypic effects. In the lactating mammary gland, levels of lipogenesis, specifically the production of medium chain fatty acids, were decreased, whereas hepatic lipogenesis was not decreased. In fact, hepatic lipogenesis was increased, and the increase may be due to compensation by a paralog of S14 called S14-R. S14-R is expressed in the liver but not the mammary gland. Importantly, S14 knockout mice did not have reduced levels of lipogenic enzymes, implying that it does not affect the transcriptional rate of those enzymes. Instead, S14 may act in the cytoplasm to affect lipogenesis.

Three major types of opioid receptors, (MOR), ␦ (DOR), and (KOR), have been cloned and characterized. Each opioid receptor exhibits a distinct pharmacological profile as well as a distinct pattern of temporal and spatial expression in the brain, suggesting the critical role of transcription regulatory elements and their associated factors. Here, we report the identification of a minimum core promoter, in the 5-flanking region of the mouse DOR gene, containing an E box and a GC box that are crucial for DOR promoter activity in NS20Y cells, a DOR-expressing mouse neuronal cell line. In vitro protein-DNA binding assays and in vivo transient transfection assays indicated that members of both the upstream stimulatory factor and Sp families of transcription factors bound to and trans-activated the DOR promoter via the E box and GC box, respectively. Furthermore, functional and physical interactions between these factors were critical for the basal as well as maximum promoter activity of the DOR gene. Thus, the distinct developmental emergence and brain regional distribution of the ␦ opioid receptor appear to be controlled, at least in part, by these two regulatory elements and their associated factors.

The main strategy of gene therapy has traditionally been focused on gene augmentation. This approach typically involves the introduction of an expression system designed to express a specific protein in the transfected cell. Both the basic and clinical sciences have generated enough information to suggest that gene therapy would eventually alter the fundamental practice of modern medicine. However, despite progress in the field, widespread clinical applications and success have not been achieved. The myriad deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders. One, derived primarily from the pioneering work of homologous recombination, is gene repair. Simply stated, the process involves targeting the mutation in situ for gene correction and a return to normal gene function.Site-specific genetic repair has many advantages over augmentation although it too is associated with significant limitations. This review outlines the advantages and disadvantages of gene correction. In particular, we discuss technologies based on chimeric RNA/DNA oligonucleotides, single-stranded and triplex-forming oligonucleotides, and small fragment homologous replacement. While each of these approaches is different, they all share a number of common characteristics, including the need for efficient delivery of nucleic acids to the nucleus. In addition, we review the potential application of a novel and exciting nonviral gene augmentation strategy-the Sleeping Beauty transposon system.