Human surfactant protein B promoter in transgenic mice: temporal, spatial, and stimulus-responsive regulation

Surfactant protein B (SP-B) is a developmentally and hormonally regulated lung protein that is required for normal surfactant function. We generated transgenic mice carrying the human SP-B promoter (-1,039/+431 bp) linked to chloramphenicol acetyltransferase (CAT). CAT activity was high in lung and immunoreactive protein localized to alveolar type II and bronchiolar epithelial cells. In addition, thyroid, trachea, and intestine demonstrated CAT activity, and each of these tissues also expressed low levels of SP-B mRNA. Developmental expression of CAT activity and SP-B mRNA in fetal lung were similar and both increased during explant culture. SP-B mRNA but not CAT activity decreased during culture of adult lung, and both were reduced by transforming growth factor (TGF)-beta(1). Treatment of adult mice with intratracheal bleomycin caused similar time-dependent decreases in lung SP-B mRNA and CAT activity. These findings indicate that the human SP-B promoter fragment directs tissue- and lung cell-specific transgene expression and contains cis-acting elements involved in regulated expression during development, fetal lung explant culture, and responsiveness to TGF-beta and bleomycin-induced lung injury.     

Tissue-specific and hormonally regulated expression of the puromycin N-acetyltransferase-encoding gene under control of the rabbit uteroglobin promoter in transgenic mice.

Transgenic mice bearing two fragments of the rabbit uteroglobin 5'-flanking region fused to the new reporter gene (pac) encoding puromycin N-acetyltransferase (PAC) showed a different pattern of expression. Transgenic lines (C0.4) harboring a 404-bp fragment (-396/+8) had a uterus-specific expression slightly inducible by estrogen, lacking detectable expression in other tissues where the uteroglobin-encoding gene is naturally expressed in rabbit. Transgenic lines (C3.2) bearing a longer fragment of 3.2-kb (-3254/+8) showed hormonally regulated expression in the uterus and the male genital tract, and detectable expression in the lung. In addition, the nonstimulated uterine expression of the transgene was higher in C0.4 lines than in C3.2 lines. It could be concluded that all sequences required for uterus-specific expression should be present within the 404-bp fragment, and that other upstream (-396) sequences are responsible for expression in the lung and male genital tract, as well as for a possible down modulation of expression in the uterus.     

Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli

A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein.     

SV40 T抗原与人U_1和U_2 snRNA基因的特异结合

利用DNA结合免疫分析证明,编码人U_1和U_2 snRNA基因的5′端区域含有与SV40 T抗原特异结合的序列。SV40 T抗原与U_2基因的亲和力大于U_1基因。DNasel足印(footprinting)分析取得与DNA结合免疫分析一致的结果。U_1和U_2基因的5′端区域含有能被T抗原所保护的,免于DNasel降解的序列。这两个基因的两条链上都含有约30bp长的DNA被T抗原所保护。U_1基因被保护的区域在-11bp—-42bp之间,而U_2基因被保护区域是在-58bp—-90bp之间。