Vitellogenin gene transcription: a relative quantitative exposure indicator of environmental estrogens.

We report the development of a quantifiable exposure indicator for measuring the presence of environmental estrogens in aquatic systems. Synthetic oligonucleotides, designed specifically for the vitellogenin gene (Vg) transcription product, were used in a reverse transcription-polymerase chain reaction (RT-PCR) protocol. This extremely sensitive and rapid method was able to detect vitellogenin gene transcription in male common carp (Cyprinus carpio) injected with 17beta-estradiol. Sequence analysis of the induced mRNA product confirmed a vitellogenin gene transcript with homology to rainbow trout and fathead minnow vitellogenin cDNA sequences. Relative levels of vitellogenin gene induction among individuals were quantified by incorporating 18S ribosomal RNA universal primers and Competimers in a PCR multiplex reaction with primers for vitellogenin. This method is more sensitive than current protocols, such as mortality, visible signs of stress, or other techniques that look for unscheduled gene expression, because it measures the appearance of primary transcripts at the nanogram level. In addition, this procedure does not sacrifice accuracy or reliability, even though the exposure to estrogen is within 1 d. This research will support the construction of regional stressor profiles, thereby providing a method for comparative environmental exposure assessment. It may also provide an in vivo screening method for potential endocrine-disrupting compounds.

Cis-active elements controlling lung cell-specific expression of human pulmonary surfactant protein B gene.

Human surfactant protein B (SPB) is a 79-amino-acid hydrophobic protein that enhances the surface active properties of pulmonary surfactant. SPB is expressed in nonciliated bronchiolar and alveolar type II cells of the respiratory epithelium, and its expression increases markedly late in gestation. In the present study, a human pulmonary adenocarcinoma cell line, H441, was used in both functional and biochemical assays to identify DNA sequences controlling lung cell-specific expression of the SPB gene. DNase I hypersensitive studies demonstrated two distinct regions of lung cell-specific hypersensitivity located proximal to the SPB promoter and within the eighth intron of the gene. To functionally define these DNA sequences, a series of plasmid vectors were constructed in which segments of the human SPB gene and 5'-flanking sequence were linked to a CAT reporter gene and assayed for expression in lung and nonlung cell lines. Whereas far upstream and intronic sequences did not contain enhancer-like elements, a 259-base pair DNA segment (base pair -218 to +41) was sufficient to support lung cell-specific expression. DNase I footprinting demonstrated that this pulmonary epithelial cell-specific promoter fragment contained five nuclear protein-binding sites, two of which bound lung cell-specific nuclear protein complexes. These results suggest that the pulmonary epithelial cell-specific expression of SPB is determined, in part, by both ubiquitous and cell type-specific protein-DNA interactions within the proximal promoter region.

Murine pulmonary surfactant SP-A gene: cloning, sequence, and transcriptional activity.

SP-A is an abundant pulmonary surfactant-associated protein whose expression is controlled in a cell- and developmental-specific manner. To analyze regulation of SP-A gene expression, the murine SP-A gene was cloned and sequenced. The murine DBA/2J gene was approximately 4.6 kb in length comprised of six exons and five introns. Three mRNAs of 3.0, 1.7, and 0.9 kb were detected by Northern blot analysis of murine lung mRNA. Expression of the SP-A mRNAs was first detected at day 15 of gestation and increased dramatically before birth. A single SP-A gene was detected in the DBA/2J mouse genome. SP-A mRNA was detected in lung but not in the gastrointestinal tract, kidney, brain, liver, or heart and was detected by in situ hybridization in bronchial and alveolar cells of the murine lung. Primer extension analysis with a primer to exon three revealed two extension products differing by 9 bp in length, suggesting two closely juxtaposed transcription initiation sites. Chimeric gene(s) containing 1.8 kb of 5' SP-A sequences and the bacterial chloramphenicol acetyltransferase gene were expressed in pulmonary adenocarcinoma cells and in HeLa cells. Expression of the murine SP-A gene is partially controlled by non-cell-selective transcriptionally active sequences.

cis-acting elements that confer lung epithelial cell expression of the CC10 gene.

To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice.

Strategies for analysis of gene expression: pulmonary surfactant proteins.

Gene transcription is regulated by the formation of protein-DNA complexes that influence the rate of specific initiation of transcription by RNA polymerase. Recent experimental advances allowing the identification of cis regulatory sequences that specify the binding of trans acting protein factors have made significant contributions to our understanding of the mechanistic complexities of transcriptional regulation. These methodologies have prompted the use of similar strategies to elucidate transcriptional control mechanisms involved in the tissue specific and developmental regulation of pulmonary surfactant protein gene expression. The purpose of this review is to describe various methodologies by which molecular biologists identify and subsequently assay regions of nucleic acids presumed to be integral in gene regulation at the level of transcription. It is well established that genes encoding surfactant proteins are subject to regulation by hormones, cytokines, and a variety of biologically active reagents. Perhaps future studies utilizing molecular tools outlined in this review will be valuable in identification of DNA sequences and protein factors required for the regulation of lung surfactant genes.

Cell type-specific transcriptional regulation of the human adenosine deaminase gene.

The relative rates of transcription of the human adenosine deaminase (ADA) gene were determined in isolated nuclei from T and B lymphoblasts and skin fibroblasts. ADA gene transcription occurs at higher rates in T cells than in B cells and fibroblasts. Relative steady state ADA mRNA levels were also determined for each cell line, and these values were found to correlate with relative rates of transcription of the gene. Therefore, the primary mechanism for control of expression of this ubiquitous enzyme is at the level of transcription. The ratios of ADA enzymatic activity to specific mRNA content were also compared between cell lines. The B lymphoblasts exhibited lower ratios than did the T lymphoblasts, suggesting that rates of protein degradation were several fold greater in B than in T lymphoblast cell lines. This finding is consistent with previous direct measurements of ADA protein turnover. Differential rates of protein turnover in B as compared to T cells provide a secondary mechanism for the regulation of ADA expression. In addition to transcription initiation being the major control mechanism of steady state ADA mRNA in all cell lines, first intron elongation pausing occurs in fibroblasts, and discrete regions of RNA polymerase II and RNA polymerase III antisense transcripts are observed in all cell lines studied.

Complete sequence and structure of the gene for human adenosine deaminase.

The nucleotide sequence of the human adenosine deaminase gene was determined. The gene was isolated in a series of overlapping lambda phage clones containing human germ line DNA. A total of 36,741 base pairs were sequenced, including 32,040 base pairs from the transcription initiation site to the polyadenylation site, 3935 base pairs of 5'-flanking DNA, and 766 base pairs of 3'-flanking DNA. The gene contains 12 exons separated by 11 introns. The exons range in size from 62 to 325 base pairs while the introns are 76-15 166 base pairs in size. The area sequenced contains 23 copies of Alu repetitive DNA and a single copy of an "O" family repeat. All but one of these repeat sequences are located in the first three introns or the 5'-flanking region. The apparent promoter region of the gene lacks the "TATA" and "CAAT" sequences often found in eucaryotic promoters and is extremely G/C rich. Contained within this region are areas homologous to other G/C-rich promoters, including six decanucleotide sequences that are highly homologous to sequences identified as functional binding sites for transcription factor Sp1.