Overexpression of CCAAT displacement protein represses the promiscuously active proximal gp91(phox) promoter.

CCAAT displacement protein (CDP) is a transcriptional repressor that restricts expression of the gp91(phox) gene to mature myeloid cells. CDP interacts with multiple sites within the -450 to +12 bp human gp91(phox) promoter, and down-regulation of CDP DNA-binding activity is required for induction of gp91(phox) transcription in mature phagocytes. Truncation of the gp91(phox) promoter to -102 to +12 bp removes 4 CDP-binding sites and reveals a promiscuous promoter activity that is active in some nonphagocytic cells. A cis-element at -90 bp is required for derepressed transcription and serves as a binding site for multiple transcriptional activators. We now report that this element also serves as a binding site for CDP. The affinity of CDP for this element is relatively weak compared with upstream CDP-binding sites within the promoter, consistent with the promiscuous transcriptional activity exhibited by the -102 to +12 bp gp91(phox) promoter fragment. Further analysis of the proximal promoter reveals an additional weak-affinity CDP-binding site centered at approximately -20 bp. Overexpression of cloned CDP represses the -102 to +12 bp gp91(phox) promoter, indicating that these proximal CDP-binding sites are functionally significant. The constellation of transcriptional activators and a repressor that interacts with the -90 bp cis-element is identical to that observed for a promoter element at -220 bp, reflecting the highly modular organization of the gp91(phox) promoter. These studies illustrate the complex interplay between transcriptional activators and a repressor that contribute to the myeloid-restricted expression of the gp91(phox) gene.

DNA-binding properties of CCAAT displacement protein cut repeats.

CCAAT displacement protein (CDP) is a transcriptional repressor that contains four distinct DNA-binding domains; a homeodomain and three cut repeats. Each DNA-binding domain of CDP was expressed as a glutathione S-transferase (GST)-fusion protein and analyzed for relative binding affinity to five CDP-binding sites within the gp91phox promoter. Each cut repeat exhibits a unique pattern of DNA-binding affinities for the five binding sites in the gp91phox promoter, suggesting that each may make a distinct contribution to the DNA-binding behavior of native CDP. Although measurement of DNA/protein complex mass indicates that an isolated cut repeat can bind DNA as a monomer, mixing of GST-cut repeat and GST-homeodomain fusion proteins enhances DNA-binding activity. Far-Western blot and two-hybrid analyses indicate, however, that the CDP domains do not directly interact. We hypothesize that GST-mediated dimerization leads to spatial juxtaposition of these DNA-binding domains, and that the resulting enhanced DNA-binding activity mimics cooperative interactions that occur between these domains in native CDP.

A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila.

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.

A genetic transformation system for Streptococcus pyogenes.

An efficient protoplast transformation system for Streptococcus pyogenes has been developed. Efficiencies of up to 7.1 x 10(6) transformants/micrograms DNA were achieved, with transformants recovered on selective media in 24-48 h. The system was characterized as to optimal protoplasting conditions, effective facilitators, dependency on concentration of transforming DNA, plasmid copy number in transformants and stability of transformants. Three isolates of S. pyogenes were used as recipients, and four plasmids were used as the transforming DNA. Growth of S. pyogenes in glycine followed by lysozyme treatment was necessary for optimal protoplast formation. The exact concentrations of these protoplasting agents which were used varied with each isolate tested. Both polyethylene glycol and dextran sulphate were efficient facilitators of transformation, at final concentrations of 10%. An inverse relationship between DNA concentration and efficiency of transformation was shown. The copy number of the AC-1 plasmid in the transformants was shown to be equivalent to that of the wild type S. pyogenes (AC-1) (one or two copies per chromosomal equivalent). Approximately 50% of the AC-1 transformants were stable after one passage on non-selective media, and 100% of those that retained the plasmid were stable for an additional twenty generations. Erythromycin resistance encoded on the AC-1 plasmid was inducible, and transformants with a constitutive mutant of the AC-1 plasmid were detected by growth on selective media. This plasmid may prove useful as a vector as it is readily transformed, expressed, and contains at least three unique restriction sites which could serve as insertion points for cloned DNA.

Preliminary studies with a live streptomycin-dependent Pasteurella multocida and Pasteurella haemolytica vaccine for the prevention of bovine pneumonic pasteurellosis.

Twelve Pasteurella-free Holstein-Friesian calves were used in a study to test the efficacy of a live streptomycin-dependent Pasteurella multocida A:3 and streptomycin-dependent Pasteurella haemolytica A1 vaccine. The calves were inoculated intramuscularly twice at 14-day intervals with either the streptomycin-dependent vaccine, containing 1 X 10(6) colony forming units/mL P. multocida and 4 X 10(8) colony forming units/mL P. haemolytica, commercial bacterin, or phosphate buffered saline. Two weeks following the second vaccination, all calves were challenged by intranasal inoculation of 10(8) TCID50/4.0 mL infectious bovine rhinotracheitis virus followed three days later by intratracheal injection with 2.3 X 10(7) colony forming units/mL of a 16 hour culture of P. multocida A:3 and 2.6 X 10(8) colony forming units/mL of an 8 hour culture of P. haemolytica A1. Seven days after challenge with Pasteurella, calves were killed for collection of tissues at necropsy. Each calf was given a score based on macroscopic and microscopic lesions. The scores for the calves receiving live vaccines were significantly lower (p less than 0.025) than those for the controls. Also, the calves receiving live vaccines had a significant (p less than 0.05) increase in the level of serum antibody to P. haemolytica. The results of this preliminary study showed that the streptomycin-dependent vaccine offered better protection than the commercial bacterin against a virulent homologous challenge.