cDNA sequence and alternative mRNA splicing of surfactant-associated protein C (SP-C) in rabbit lung.
An 784 base pair (bp) copy DNA (cDNA) for the low molecular weight hydrophobic surfactant-associated protein C (SP-C) has been isolated from a lambda gt11 cDNA library constructed from fetal rabbit lung mRNA. The cDNA, which coded for a 193 amino-acid proprotein with 6 bp 5' and 193 bp 3' untranslated segments, possesses considerable nucleic acid and predicted amino-acid homology with previously reported SP-C cDNAs. The predicted amino-acid sequence of the 35 amino-acid mature polypeptide shares 94-97% identity with human, rat and mouse SP-C and is 88-91% homologous to the mature proteins from bovine, porcine and canine lung. The last 12 amino acids of mature SP-C are highly hydrophobic and invariant. Alignment of the rabbit and human nucleic acid sequences required introduction of a 27 bp gap in the rabbit sequence at a site corresponding to the exon-intron junction of the 5th exon of the human genomic sequence. Since previous studies have identified differential splicing at the 5' and 3' ends of the human 5th exon, we investigated the potential existence of alternative splicing of rabbit SP-C mRNA. Reverse transcription (RT) of total RNA followed by polymerase chain reaction (PCR) was used to establish the relative abundance of alternative splicing products from fetal and adult lung and from rabbit kidney, placenta and liver. The relative abundance of the 250, 280 and 350 bp bands observed was the same in lung and other tissues. PCR amplification of genomic rabbit DNA indicated that the 350 bp fragment corresponds to the unspliced nascent transcript. The lack of developmental or tissue-specific abundance patterns implies the absence of secondary influences on SP-C mRNA polymorphism. Indeed, free energy of formation calculations predicted the presence of hairpin structures favouring formation of the more abundant 250 bp form. These observations plus the absence of any effect of alternative splicing on SP-C protein structure led us to conclude a physiological role is unlikely.
Connelly
I
I
MRC Group in Fetal and Neonatal Health and Development, University of Western Ontario, London, Canada.
Possmayer
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D005091:Exons; D005333:Fetus / Q000378:metabolism; D015723:Gene Library; D008168:Lung / Q000378:metabolism*; D008969:Molecular Sequence Data; D011510:Proteolipids / Q000096:biosynthesis / Q000235:genetics*; D011663:Pulmonary Surfactants / Q000096:biosynthesis / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid
D011510:Proteolipids; D011663:Pulmonary Surfactants; D012333:RNA, Messenger; D004247:DNA