Expression of the Moraxella catarrhalis UspA1 protein undergoes phase variation and is regulated at the transcriptional level.

The UspA1 protein of Moraxella catarrhalis has been shown to function as an adhesin that mediates adherence to human epithelial cell lines in vitro (E. R. Lafontaine, L. D. Cope, C. Aebi, J. L. Latimer, G. H. McCracken, Jr., and E. J. Hansen, J. Bacteriol. 182:1364-1373, 2000). In the present study, cell lysates prepared from individual colonies of several M. catarrhalis wild-type strains were analyzed by Western blot analysis using monoclonal antibodies (MAbs) specific for the UspA1 protein. Expression of UspA1 was shown to exhibit phase variation that was correlated with both adherence ability in vitro and the number of guanine (G) residues contained within a homopolymeric [poly(G)]tract located upstream of the uspA1 open reading frame (ORF). Nucleotide sequence analysis revealed that isolates expressing relatively high levels of UspA1 had 10 G residues in their uspA1 poly(G)tracts, whereas isolates that expressed much lower levels of UspA1 had 9 G residues. This poly(G) tract was located 30 nucleotides (nt) upstream of the uspA1 ORF and 168 nt downstream of the uspA1 transcriptional start site. Primer extension experiments, RNA slot blot analysis, and cat reporter constructs were used to demonstrate that M. catarrhalis isolates with 10 G residues in their uspA1 poly(G) tracts expressed two-to threefold more uspA1 mRNA than did isolates which had 9 G residues in their poly(G)tracts. Northern hybridization analysis revealed that an intact uspA1 mRNA was readily detectable in RNA from M. catarrhalis isolates that had 10 G residues in their uspA1 poly(G) tracts, whereas no full-length uspA1 mRNA was observed in isolates whose poly(G)tracts contained 9 G residues. M. catarrhalis strain O35E uspA1 genes that contained wild-type and mutated poly(G) tracts were expressed in Haemophilus influenzae to demonstrate that the length and composition of the poly(G)tract affected expression of UspA1.

Transcriptional regulation of the chicken CYP2H1 gene. Localization of a phenobarbital-responsive enhancer domain.

The mechanism by which the drugs phenobarbital and 2-allyl-2-isopropylacetamide induce levels of chicken cytochrome P-450 (CYP) mRNAs has been investigated in primary hepatocyte cultures from 17-day-old chick embryos. It has been demonstrated that three CYP mRNAs of 3.5, 2.5, and 2.2 kilobases (kb) are strongly induced by phenobarbital in primary hepatocytes, as found previously in chick embryo liver in ovo (Hansen, A. J., Elferink, L. A., and May, B. K. (1989) DNA (NY) 8, 179-191), and that, at least for the 3.5-kb mRNA, this is predominantly a result of enhanced transcription of the corresponding gene, CYP2H1. Transient transfection assays were carried out in primary cultures using constructs containing different lengths of CYP2H1 gene 5'-flanking sequence fused to the reporter chloramphenicol acetyl-transferase (CAT) gene. These experiments established that cis-acting elements located in the first 0.5 kb of the CYP2H1 gene 5'-flanking region direct high basal expression of the CAT gene, but do not mediate phenobarbital inducibility. When constructs containing more than 1.1 kb of CYP2H1 gene 5'-flanking sequence were examined, phenobarbital induction of CAT expression was observed, and a drug-responsive domain between positions -5.9 and -1.1 kb was identified. This domain has the properties of an enhancer, since it is able to confer phenobarbital responsiveness to the enhancerless SV40 promoter when tested in either orientation or at different distances from the promoter. The enhancer domain also responds to 2-allyl-2-isopropylacetamide, but whether the action of the two drugs is mediated by a single nuclear receptor interacting with common DNA elements in the domain remains to be established.