SP-C Gene Research Articles

The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis.

We used the lung epithelial cell-specific surfactant protein B (SPB) gene promoter as a model with which to investigate mechanisms involved in transcriptional control of lung-specific genes. In a previous study, we showed that the SPB promoter specifically activated expression of a linked reporter gene in the continuous H441 lung cell line and that H441 nuclear proteins specifically protected a region of this promoter from bp -111 to -73. In this study, we further show that this region is a complex binding site for thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3 (HNF-3). Whereas TTF-1 bound two highly degenerate and closely spaced sites, HNF-3 proteins bound a TGT3 motif (TGTTTGT) that is also found in several liver-specific gene regulatory regions, where it appears to be a weak affinity site for HNF-3. Point mutations of these binding sites eliminated factor binding and resulted in significant decreases in transfected SPB promoter activity. In addition, we developed a cotransfection assay and showed that a family of lung-specific gene promoters that included the SPB, SPC, SPA, and Clara cell secretory protein (CCSP) gene promoters were specifically activated by cotransfected TTF-1. We conclude that TTF-1 and HNF-3 are major activators of lung-specific genes and propose that these factors are involved in a general mechanism of lung-specific gene transcription. Importantly, these data also show that common factors are involved in organ-specific gene expression along the mammalian foregut axis.

The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis.

We used the lung epithelial cell-specific surfactant protein B (SPB) gene promoter as a model with which to investigate mechanisms involved in transcriptional control of lung-specific genes. In a previous study, we showed that the SPB promoter specifically activated expression of a linked reporter gene in the continuous H441 lung cell line and that H441 nuclear proteins specifically protected a region of this promoter from bp -111 to -73. In this study, we further show that this region is a complex binding site for thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3 (HNF-3). Whereas TTF-1 bound two highly degenerate and closely spaced sites, HNF-3 proteins bound a TGT3 motif (TGTTTGT) that is also found in several liver-specific gene regulatory regions, where it appears to be a weak affinity site for HNF-3. Point mutations of these binding sites eliminated factor binding and resulted in significant decreases in transfected SPB promoter activity. In addition, we developed a cotransfection assay and showed that a family of lung-specific gene promoters that included the SPB, SPC, SPA, and Clara cell secretory protein (CCSP) gene promoters were specifically activated by cotransfected TTF-1. We conclude that TTF-1 and HNF-3 are major activators of lung-specific genes and propose that these factors are involved in a general mechanism of lung-specific gene transcription. Importantly, these data also show that common factors are involved in organ-specific gene expression along the mammalian foregut axis.

Transcriptional Elements from the Human SP-C Gene Direct Expression in the Primordial Respiratory Epithelium of Transgenic Mice

Transgenic animals bearing a chimeric gene containing 5′-flanking regions of the human surfactant protein C (SPC) gene ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene were analyzed by in situ hybridization histochemistry to determine the temporal and spatial distribution of transgene expression during organogenesis of the murine lung. Ontogenic expression of the SP-C-CAT gene was compared to that of the endogenous SP-C gene and to the Clara cell CC10 gene. High levels of SP-C-CAT expression were observed as early as Day 10 of gestation in epithelial cells of the primordial lung buds. Low levels of endogenous SP-C mRNA were detected a day later, but only in the more distal epithelial cells of the newly formed, primitive, lobar bronchi. On Gestational Days 13 through 16, transcripts for both the endogenous and chimeric gene were restricted to distal epithelial elements of the branching bronchial tubules and were no longer detected in the more proximal regions of the bronchial tree. Although high levels of SP-C-CAT expression were maintained throughout organogenesis, endogenous SP-C expression increased dramatically on Gestational Day 15, coincident with acinar tubule differentiation at the lung periphery. Low levels of endogenous CC10, expression were detected by Gestational Day 16 in both lobar and segmental bronchi. By the time of birth, CC10 transcripts were expressed at high levels in the trachea and at all levels of the bronchial tree; endogenous SP-C mRNA was restricted to epithelial cells of the terminal alveolar saccules; and SP-C-CAT expression was now detected in both alveolar and bronchiolar epithelial cells. These results indicate that (1) cis -acting regulatory elements of the human SP-C gene can direct high levels of foreign gene expression to epithelial cells of the embryonic mouse long; (2) expression of the human SP-C-CAT chimeric gene is developmentally regulated, exhibiting a morphogenic expression pattern similar, but not identical, to that of the endogenous murine SP-C gene; (3) the embryonic expression of endogenous SP-C and chimeric SP-C-CAT transcripts identifies progenitor cells of the distal respiratory epithelium; and (4) differentiation of bronchial epithelium is coincident with loss of SP-C expression and subsequent acquisition of CC10, expression in proximal regions of the developing bronchial tubules.

Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs

Pages L634–L644: Vijayakumar Boggaram and Ramgopal K. Margana. “Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs.” Page L634, left-hand column, in the abstract, the sentence beginning on line 22 should read: Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase chain reaction-amplification of genomic DNA with oligonucleotide primers flanking the insertional sequence and sequence analysis of amplified DNA showed that SP-C mRNAs are produced by alternative use of 3' splice sites of intron 5 of SP-C gene. Page L634, left-hand column, paragraph 2, line 4 should read: SP-A [relative molecular weight (Mr) ≈35,000] and SP-D (Mr≈43,000) are hydrophilic proteins that are similar in having collagenous amino-terminus domain and noncollagenous lectinlike carboxy-terminus domain. SP-B (Mr ap8,000) and SP-C (Mr ap5,000) are hydrophobic proteins derived from larger precursor proteins by amino- and carboxy-terminus proteolytic processing.

Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs

Pages L634–L644: Vijayakumar Boggaram and Ramgopal K. Margana. “Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs.” Page L634, left-hand column, in the abstract, the sentence beginning on line 22 should read: Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase chain reaction-amplification of genomic DNA with oligonucleotide primers flanking the insertional sequence and sequence analysis of amplified DNA showed that SP-C mRNAs are produced by alternative use of 3' splice sites of intron 5 of SP-C gene. Page L634, left-hand column, paragraph 2, line 4 should read: SP-A [relative molecular weight ( Mr)≈35,000] and SP-D ( Mr ≈43,000) are hydrophilic proteins that are similar in having collagenous amino-terminus domain and noncollagenous lectinlike carboxy-terminus domain. SP-B ( Mr ≈8,000) and SP-C ( Mr ≈5,000) are hydrophobic proteins derived from larger precursor proteins by amino- and carboxy-terminus proteolytic processing.

Surfactant protein C: a review of its unique properties and metabolism.

Traditionally, our thinking about surfactant proteins has centered around their effects on the biophysical properties of surfactant phospholipids. It is now apparent that the three major surfactant proteins (SP-A, SP-B, and SP-C) are a biochemically and functionally diverse group of mammalian peptides. Accumulated data suggest that they have roles beyond modulation of alveolar surface tension. SP-C is a 33-35 amino acid peptide found in organic extracts of pulmonary surfactant. In part, because of its extreme hydrophobicity, a full understanding of SP-C is presently incomplete. Progress to date has included evaluation of the biophysical properties and investigations of the SP-C gene, including studies of the SP-C promoter. This review describes the unique structural and functional properties of the SP-C molecule and summarizes available data on its molecular biology and metabolism. Studies from literature show that SP-C represents a physiologically important peptide with novel structural properties; namely, extreme hydrophobicity, an alpha-helical membrane spanning region, and a unique posttranslational modification: palmitoylation. From data on similarly modified proteins, we propose that the properties of SP-C, including the covalent addition of palmitic acid, render it capable of being targeted to and interacting with specific cell membranes. A complete understanding of SP-C, especially with regard to its metabolism and function, may require a reorientation of our thinking to consider SP-C as a membrane peptide and not just as a "surfactant protein."

Hormonal Control of the Surfactant System in Fetal Lung

The synthesis of surfactant glycerophospholipids and proteins is under multifactorial control and is regulated by a number of hormones and factors, including glucocorticoids, prolactin, insulin, growth factors, estrogens, androgens, thyroid hormones and catecholamines acting through beta-adrenergic receptors, and cAMP. In studies with human fetal lung in organ culture, glucocorticoids, in combination with prolactin and/or insulin, were found to increase the rate of lamellar body PC synthesis and increase the molar ratio of surfactant PG to PI to a value similar to that of surfactant secreted by the human fetal lung at term. Recognition of the potential importance of the surfactant proteins SP-A, SP-B, and SP-C in the reduction of alveolar surface tension and in endocytosis and reutilization of secreted surfactant by type II cells has stimulated rapid advancement of knowledge concerning the structures of these proteins and their genes, as well as their developmental and hormonal regulation in fetal lung tissue. The genes encoding the surfactant proteins are expressed in a lung-specific manner and appear to be regulated independently during fetal development. SP-A gene expression is initiated in fetal lung tissue after 75-85% of gestation is completed in all mammalian species studied to date. In the human fetus, however, expression of the SP-B and SP-C genes is detectable prior to mid-gestation. In situ hybridization studies of human lung tissue indicate that the SP-A gene is expressed only in type II cells, whereas SP-B gene expression is detectable in bronchioalveolar epithelial cells as well. Cyclic AMP and glucocorticoids have pronounced effects on the regulation of SP-A gene expression in human and rabbit fetal lung in culture. In human fetal lung in vitro, the effects of cAMP are primarily at the level of gene transcription. By contrast, glucocorticoids have stimulatory effects on SP-A gene transcription and inhibitory effects on SP-A mRNA stability. Furthermore, the combined effects of cAMP and glucocorticoids on SP-A gene transcription in human fetal lung in vitro are synergistic. Glucocorticoids appear to be of primary importance in the regulation of the genes encoding SP-B and SP-C. Elucidation of the molecular mechanisms involved in the regulation of expression of the surfactant protein genes in developing fetal lung will be of fundamental importance to our understanding of the developmental and tissue-specific regulation of eukaryotic gene expression.

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.

9 Hormonal and developmental regulation of pulmonary surfactant synthesis in fetal lung

Pulmonary surfactant, a unique developmentally regulated, phospholipid-rich lipoprotein, is synthesized by the type II cells of the pulmonary alveolus, where it is stored in organelles termed lamellar bodies. The principal surface-active component of surfactant, dipalmitoylphosphatidylcholine, a disaturated form of phosphatidylcholine, acts in concert with the surfactant-associated proteins to reduce alveolar surface tension. Relatively large amounts of phosphatidylglycerol also are present in lung surfactants of a number of species, including man. The role of phosphatidylglycerol in surfactant function has not been elucidated; however, its presence in increased amounts in pulmonary surfactant is correlated with enhanced fetal lung maturity. Surfactant glycerophospholipid synthesis in fetal lung tissue is regulated by a number of hormones and factors, including glucocorticoids, prolactin, insulin, oestrogens, androgens, thyroid hormones, and catecholamines acting through cyclic AMP. In studies with human fetal lung in organ culture, we have observed that glucocorticoids, in combination with prolactin and/or insulin, increase the rate of lamellar body phosphatidylcholine synthesis and alter lamellar body glycerophospholipid composition to one reflective of surfactant secreted by the human fetal lung at term. Four surfactant-associated proteins, SP-A, SP-B, SP-C and SP-D, have recently been characterized. Recognition of their potential importance in the reduction of alveolar surface tension and in endocytosis and reutilization of secreted surfactant by type II cells has stimulated rapid advancement of knowledge concerning the structures of the surfactant proteins and their genes, as well as their developmental and hormonal regulation in fetal lung tissue. The genes encoding SP-A, SP-B and SP-C are expressed in a cell-specific manner and are independently regulated in fetal lung tissue during development. SP-A gene expression occurs exclusively in the type II cell and is initiated after 75% of gestation is complete. In the human fetus, expression of the SP-B and SP-C genes is detectable much earlier in development than SP-A, before the time of appearance of differentiated type II cells. It is apparent from studies using human and rabbit fetal lung in culture that cyclic AMP and glucocorticoids serve important roles in the regulation of SP-A gene expression. While the effects of cyclic AMP are exerted primarily at the level of gene transcription in human fetal lung tissue, glucocorticoids have stimulatory effects on SP-A gene transcription and inhibitory effects on SP-A mRNA stability. In addition, cyclic AMP and glucocorticoids act synergistically to increase SP-A gene transcription in human fetal lung in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

High frequency conjugal transfer of tylosin genes and amplifiable DNA in Streptomyces fradiae.

Genes encoding enzymes for tylosin biosynthesis, genes involved in the expression of resistance to tylosin (Tyl), hygromycin B (Hm), chloramphenicol (Cm), and mitomycin C (MC), and a single copy of an amplifiable unit of DNA (AUD) were jointly transferred at very high frequencies by conjugation from several different Streptomyces fradiae strains to S. fradiae JS85, a mutant defective in many or possibly all tylosin biosynthetic reactions and containing a multiple tandem reiteration of the AUD. No recombination was observed between nar, rif and spc genes in conjugal matings, but recombination was observed between these genes after protoplast fusion. Tylosin biosynthetic genes were transferred at a much lower frequency to S. fradiae JS87, another mutant defective in many or all tylosin biosynthetic reactions, but deleted for the AUD and other DNA sequences. These findings suggest that tylosin structural genes, several genes encoding antibiotic resistance determinants, and amplifiable DNA are present on a self-transmissible element that does not mobilize chromosomal genes, and that JS85 and JS87 contain deletions, and JS85 an amplification, of overlapping portions of this element.

Organization of ribosomal protein genes of Escherichia coli as analyzed by polar insertion mutations.

Several mutants of lambdaspc1 and lambdafus3 have been isolated carrying DNA insertion elements that were selected for their ability to reduce the expression of the spc gene. The sizes and locations of the insertions on the phage genomes were determined by heteroduplex analysis. They were found to be located at different positions in the Spc transcription unit. The effect of these insertions on the expression of the ribosomal protein genes carried by these phages in ultraviolet light-irradiated bacteria was investigated. The insertions at intermediate positions in the transcription unit reduced the expression of some of the genes in the unit but not others. Assuming that the genes whose expressions were reduced are distal to the insertion, it was possible to determine the relative position of most of the genes in the unit. The results indicate the order of genes in the Spc transcription unit is: promoter, L14, L24, L5, S14, S8, L6, L18, (S5, L15, L30).

Deletions of ribosomal protein genes in Escherichia coli merodiploids heterozygous for resistance to streptomycin and spectinomycin.

In a merodiploid strain of Escherichia coli heterozygous for the ribosomal protein genes spc and str, deletions were observed preventing the expression of either gene but permitting the expression of the other. This suggests that the spc and str genes are in separate transcriptional units.

Studies on the physical linkage of antibiotic resistance markers to ribosomal RNA genes in Bacillus subtilis

The profile of streptomycin-, erythromycin-, bryamycin- (thiostrepton), spectinomycin and lincomycin-resistance transforming activity has been compared with the distribution of the ribosomal RNA genes on methylated albumin kieselguhr columns. We have found that the str, ery, spc and lin genes showed different methylated albumin kieselguhr profiles from the rRNA genes and are not linked at the molecular weight of DNA employed in these studies (4.5 × 10 6 single-strand molecular weight). Thus, these genes are different from rRNA cistrons, and the r-proteins which they presumably code for are not formed by the translation of rRNA templates. Only the bry gene exhibited a methylated albumin kieselguhr column distribution similar to that of the rRNA genes. However, mercuric cesium sulfate isopycnic centrifugation of rRNA- bry DNA hybrids failed to demonstrate linkage between rRNA genes and the bry marker, at this level.

A ribosomal ambiguity mutation

The existence of a genetic determinant ram ‡ ‡ Abbreviations used: for the designation of genetic markers the proposals of Demerec, Adelberg Clark & Hartman (1966) have been followed and abbreviations correspond generally to those of Taylor & Trotter (1967) with the addition of apt for the speotinomycin gene. Other abbreviations are the following: CSD, conditionally streptomycin dependent (Gorini &Kataja, 1984u); Arg-CSD, streptomycin dependent under condition of absence of arginine; Sm. streptomycin; Sp, speotinomyoin; Phe, phenylalanine; De, isoleucine; Ser, serine; His, histidine; Pro, proline; aromix, mixture of growth factors required by aroE mutants which are blocked in the dehydrosbikimate reductase (Pittard & Wallace, 1966); poly-I?, polyuridine; poly-C, polycytidine. for a 30 s ribosomal component controlling translation ambiguity has been demonstrated. It is located in the strA (streptomycin sensitivity) region and is 99% cotransducible with the spc (spectinomycin sensitivity) gene. In vitro, ram mutant ribosomes misread extensively and by interchanging the subunits of ram and ram + ribosomes it is shown that misreading is a property of the 30 s subunit. In vivo, the ram mutation confers on the cell the ability to suppress all three nonsense codons. Streptomycin induces a similar generalized ambiguity phenotypically both in vitro and in vivo. The effects of ram and of streptomycin are additive. Ambiguity caused by ram, by streptomycin and by their combination is antagonized in vivo and in vitro by additional mutations at the strA locus. Thus the strA40 (resistant competent) and strA1 (resistant incompetent) alleles increasingly restrict ribosomal (as well as tRNA) suppression and return to normal the reduced growth rate produced by ram mutation in a wild-type strA + strain. Growth is inhibited completely in a ram1, strA + strain by sublethal doses of streptomycin and in ram1, strA40 strains by higher doses, while strain ram1, strA1 is indifferent to streptomycin. The bactericidal action of the drug is unrelated to these effects.