Biomedical subjects

M D Bruno

Publications and source records attributed to M D Bruno.

21 records · Page 2Linked to original sources

PubMed1991

A portion of the human surfactant protein A (SP-A) gene locus consists of a pseudogene.

SP-A is the most abundant, surfactant-associated protein isolated from lung lavage. Genomic blot analysis of total human cellular DNA with SP-A cDNA demonstrated the presence of multiple hybridizing fragments that are not accounted for by available SP-A gene sequences. In this report, we have cloned and characterized human genomic DNA fragments that account for some of the other hybridizing fragments. These clones contain nucleotide sequences that are highly homologous to the fourth intron and fifth exon of the human SP-A gene. Sequences upstream from these SP-A-like sequences are not detectable by Northern blot hybridization of SP-A-expressing cells and the SP-A-like sequences contain premature stop codons, consistent with the interpretation that these clones represent an SP-A pseudogene. Restriction fragments consistent with this pseudogene and the functional SP-A gene are present in a human chromosome 10 genomic library made from a single chromosome, showing that the functional SP-A gene and the pseudogene are syntenic.

Bacteriophage lambda↗

PubMed1990

Structure and expression of the pulmonary surfactant protein SP-C gene in the mouse.

Genomic and cDNA clones encoding the murine pulmonary surfactant protein SP-C were isolated and sequenced to provide the primary amino acid sequence of the murine SP-C polypeptide and the structural organization of the SP-C gene locus. Murine SP-C is encoded by a single locus spanning approximately 3.2 kilobases of genomic DNA. The gene is composed of six exons and five introns, and its structure is closely related to the human SP-C gene. The primary transcript for the murine SP-C preprotein of Mr = 21,000 has an overall nucleotide identity of 80% with the coding sequence of the human precursor. The active airway peptide of 33-35 amino acids was even more closely related to the human SP-C airway peptide, sharing 95% identity at the amino acid level. The most significant structural difference between the murine and human SP-C genes was an increased size of the first intron in the murine SP-C gene. A single mRNA encoding murine SP-C of 0.8 kilobase was detected only in lung tissue, and SP-C mRNA was more abundant in neonatal and postnatal lung tissue than in fetal lung tissue. The nucleotide sequence of the promoter and 5'-flanking regions of the murine SP-C gene shared considerable identity with the human SP-C gene. Transgenic mice bearing a chimeric gene composed of the 5'-flanking and promoter regions of the human SP-C gene and the bacterial chloramphenicol acetyltransferase reporter gene were generated, demonstrating that lung-specific and developmental expression was conferred by these 5'-sequences. The chimeric gene was selectively expressed in preparations of respiratory epithelial cells, but was not expressed in alveolar macrophages isolated from the transgenic mice. The shared structural organization and developmental and tissue-specific expression of the SP-C gene between the mouse and human demonstrate considerable phylogenetic conservation of this gene and protein.

Amino Acid Sequence↗

PubMed1990

Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice.

Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5'-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.

Animals↗