Abstract
Expression of the rabbit pulmonary surfactant protein A (SP-A) gene is lung-specific, occurs primarily in type II cells, and is developmentally regulated. We previously identified two E-box-like enhancers, termed the distal binding element (DBE) and proximal binding element (PBE), in the 5'-flanking region of the rabbit SP-A gene. In the present study, the PBE was used to screen a rabbit fetal lung cDNA expression library; a cDNA insert was isolated which is highly similar in sequence to human upstream stimulatory factor 1 (hUSF1). By use of reverse transcription polymerase chain reaction, two isoforms of rabbit USF1 (rUSF1) mRNAs were identified in fetal rabbit lung and other tissues. The levels of rUSF1 mRNAs reach a peak in fetal rabbit lung at 23 days gestation, in concert with the time of initiation of SP-A gene transcription. Binding complexes of nuclear proteins obtained from fetal rabbit lung tissue and isolated type II cells with the DBE and PBE were supershifted by the addition of anti-rUSF1 IgG. Binding activity was enriched in type II cells compared with lung fibroblasts. Overexpression of rUSF1s in A549 adenocarcinoma cells positively regulated SP-A promoter activity of cotransfected reporter gene constructs. It is suggested that rUSF1s, which bind to two E-box elements in the SP-A gene 5'-flanking region, may serve a key role in the regulation of SP-A gene expression in pulmonary type II cells.
Highlights
Surfactant, a developmentally regulated lipoprotein produced by pulmonary type II cells, acts to reduce alveolar surface tension and prevent atelectasis; surfactant production is initiated in fetal lung only after ϳ75% of gestation is completed
A first strand cDNA synthesis kit was used with 50 g of total RNA isolated from lung tissues of fetal rabbits of 21, 23, 25, and 28 days gestation as templates for reverse transcription of rabbit USF1 (rUSF1) cDNAs using the primer 5 (Fig. 1B and Table I), which hybridizes to the 967–987 nucleotide region of rUSF1b mRNA
Isolation of the Rabbit USF1 cDNA Clones—To isolate cDNA clones encoding proteins that bind to E-box motifs of the distal binding element (DBE) and proximal binding element (PBE) in the 5Ј-flanking region of the rabbit surfactant protein A (SP-A) gene, a gt11 cDNA expression library constructed from mRNA isolated from 24-day fetal rabbit lung tissue was screened using a 32P-labeled double-stranded oligonucleotide corresponding to the PBE
Summary
Cloning of a cDNA Insert Encoding a PBE-binding Protein—First strand cDNA was synthesized from poly(A)ϩ RNA isolated from 24-day fetal rabbit lung tissues using random hexanucleotides and was used to generate second strand cDNA using a cDNA synthesis kit (Pharmacia Biotech Inc., You-Prime cDNA synthesis kit). A phoretic mobility shift assay; GST, glutathione S-transferase; hGH, human growth hormone; hUSF1, human upstream stimulatory factor 1; PBE, proximal binding element; PCR, polymerase chain reaction; RT, reverse transcription; rUSF1, rabbit upstream stimulatory factor 1.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
