The major apoprotein of rabbit pulmonary surfactant. Elucidation of primary sequence and cyclic AMP and developmental regulation.
The major apoprotein of rabbit pulmonary surfactant. Elucidation of primary sequence and cyclic AMP and developmental regulation.
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DOI：
10.1016/s0021-9258(18)69159-8
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发表时间：
1988-02
期刊：
影响因子：
--
通讯作者：
V. Boggaram;K. Qing;C. Mendelson
中科院分区：
文献类型：
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作者：
V. Boggaram;K. Qing;C. Mendelson
兔肺表面活性物质的主要脱辅基蛋白是一种发育和激素调节的唾液酸糖蛋白，Mr 与 29,000-36,000 (SP 29-36) 一致。在本研究中，使用特异性抗体从噬菌体 lambda gt11 的胎兔肺 cDNA 文库中分离出 SP 29-36 的克隆 cDNA 插入片段。分离出两种大小为 1.9 和 3.0 kb 的 cDNA，它们与两种 2.0 和 3.0 kb 的 mRNA 互补，这两种 mRNA 的不同主要在于其 3'-非翻译区的长度。 2.0-kb mRNA 的丰度大约是 3.0-kb mRNA 的 5 倍。用多种限制性内切酶切割的兔基因组 DNA 的 Southern 杂交分析结果表明，这两种 mRNA 是由单个基因编码的。这两种 mRNA 似乎仅在兔肺组织中表达，并且在体内和体外发育过程中受到协调调节；可杂交的 SP 29-36 mRNA 在妊娠第 26 天首次在兔肺组织中检测到，在第 31 天增加到最大值，并在出生后略有下降。 3.0-kb cDNA 由 57 个核苷酸的 5'-非翻译区、一个 741 个核苷酸的开放阅读框和一个 2,165 个核苷酸的 3'-非翻译区（包含三个聚腺苷酸加成信号）组成。 Poly(A) 添加信号的最多 5' 用于合成 2.0-kb SP 29-36 mRNA，而最多 3' 用于合成 3.0-kb mRNA。 3.0-kb cDNA的开放阅读框编码247个氨基酸的蛋白质，与狗和人肺表面活性剂的主要脱辅基蛋白高度同源。 SP 29-36 cDNA 用于评估二丁酰环 AMP (Bt2cAMP) 对器官培养中胎兔肺组织中该 mRNA 水平的影响。 Bt2cAMP 引起 SP 29-36 mRNA 的诱导，早在其添加到培养基中 2 小时后即可检测到。当放线菌酮在培养基中存在时间大于或等于 4 小时时，Bt2cAMP 的这种诱导作用被阻断。放线菌酮处理还降低了对照外植体中 SP 29-36 mRNA 的水平；从培养基中去除放线菌酮后，这种对对照和 Bt2cAMP 处理的外植体的抑制作用在 12 小时内被逆转。这些发现表明，不稳定的蛋白因子介导 SP 29-36 基因的转录及其由 Bt2cAMP 的诱导。
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.