The major apoprotein of rabbit pulmonary surfactant. Elucidation of primary sequence and cyclic AMP and developmental regulation.
The major apoprotein of rabbit pulmonary surfactant is a developmentally and hormonally regulated sialoglycoprotein, Mr congruent to 29,000-36,000 (SP 29-36). In the present study, specific antibodies were used to isolate cloned cDNA inserts for SP 29-36 from a fetal rabbit lung cDNA library in bacteriophage lambda gt11. Two species of cDNA of 1.9 and 3.0 kilobases (kb) in size were isolated that are complementary to two species of mRNA of 2.0 and 3.0 kb which differ primarily in the lengths of their 3'-untranslated regions. The 2.0-kb species of mRNA is approximately 5 times more abundant than the 3.0-kb mRNA. The results of Southern hybridization analysis of rabbit genomic DNA cut with a number of restriction enzymes are indicative that the two mRNA species are encoded by a single gene. The two mRNA species appear to be expressed only in rabbit lung tissue and are coordinately regulated during development in vivo and in vitro; hybridizable SP 29-36 mRNA is first detectable in rabbit lung tissue on day 26 of gestation, increases to a maximum on day 31, and declines somewhat after birth. The 3.0-kb cDNA is comprised of 57 nucleotides of 5'-untranslated region, an open reading frame of 741 nucleotides, and a 3'-untranslated region of 2,165 nucleotides that contains three poly(A)-addition signals. The most 5' of the poly(A) addition signals is utilized in synthesis of the 2.0-kb SP 29-36 mRNA, while the most 3' is utilized in synthesis of the 3.0-kb mRNA. The open reading frame of the 3.0-kb cDNA encodes a protein of 247 amino acids which is highly homologous to the major apoproteins of dog and human pulmonary surfactant. The SP 29-36 cDNAs were utilized to evaluate the effects of dibutyryl cyclic AMP (Bt2cAMP) on the levels of this mRNA in fetal rabbit lung tissue in organ culture. Bt2cAMP caused an induction of SP 29-36 mRNA that was detectable as early as 2 h after its addition to the medium. This inductive effect of Bt2cAMP was blocked when cycloheximide was also present in the medium for greater than or equal to 4 h. Cycloheximide treatment also reduced the levels of SP 29-36 mRNA in control explants; this inhibitory effect on control and Bt2cAMP-treated explants was reversed within 12 h of the removal of cycloheximide from the medium. These findings suggest that a labile protein factor mediates the transcription of the SP 29-36 gene and its induction by Bt2cAMP.
Boggaram
V
V
Department of Biochemistry, University of Texas Health Science Center, Dallas 75235.
Qing
K
K
Mendelson
C R
CR
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001059:Apoproteins / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004285:Dogs; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008168:Lung / Q000254:growth & development*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D013045:Species Specificity
D001059:Apoproteins; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D012333:RNA, Messenger; C046616:pulmonary surfactant apoprotein; D003994:Bucladesine; D003513:Cycloheximide; D000242:Cyclic AMP; D004262:DNA Restriction Enzymes