Xiong F, Liu D, Elsholtz H P, Hew C L
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Mol Endocrinol. 1994 Jun;8(6):771-81. doi: 10.1210/mend.8.6.7935492.
The salmon pituitary expresses two distinct gonadotropins, gonadotropin I (GTHI) and gonadotropin II (GTHII). These two hormones are synthesized in distinct pituitary cells and secreted at different stages during the reproductive cycle. To study the transcriptional regulation of the hormone-specific beta-subunit of GTHII (sGTHII beta) gene, approximately 3.5 kilobases of the 5'-flanking region was characterized and sequenced. The pituitary specificity of sGTHII beta was examined by analyzing sGTHII beta promoter activity in homologous primary pituitary cells derived from spawning rainbow trout and in a collection of heterologous cell lines. Various lengths of the 5'-flanking region of the sGTHII beta gene were ligated into a vector encoding the bacterial chloramphenicol acetyltransferase (CAT) gene, and the resulting sGTHII beta/CAT chimeric constructs were analyzed using transient expression systems. Several constructs (-3500CAT, -1260CAT, -563CAT, and -39CAT) displayed readily detectable CAT activity in the pituitary cells derived from spawning male and female rainbow trout. In contrast, three of the constructs (-3500CAT, -1260CAT, and -563CAT) were expressed only at background levels in a variety of heterologous cell lines, suggesting that the 5'-flanking sequence of sGTHII beta contains information dictating its pituitary specificity. A silencer sequence (-95 to -35, pSil) was identified, which might function to repress sGTHII beta gene expression in the nongonadotropes or in the gonadotropes at developmental stages that precede final maturation and spawning.
鲑鱼垂体表达两种不同的促性腺激素,即促性腺激素I(GTHI)和促性腺激素II(GTHII)。这两种激素在不同的垂体细胞中合成,并在生殖周期的不同阶段分泌。为了研究GTHII激素特异性β亚基(sGTHIIβ)基因的转录调控,对约3.5千碱基的5'-侧翼区域进行了特征分析和测序。通过分析来自产卵虹鳟的同源原代垂体细胞和一系列异源细胞系中的sGTHIIβ启动子活性,检测了sGTHIIβ的垂体特异性。将sGTHIIβ基因5'-侧翼区域的不同长度片段连接到编码细菌氯霉素乙酰转移酶(CAT)基因的载体中,并使用瞬时表达系统分析所得的sGTHIIβ/CAT嵌合构建体。几个构建体(-3500CAT、-1260CAT、-563CAT和-39CAT)在来自产卵雌雄虹鳟的垂体细胞中表现出易于检测到的CAT活性。相比之下,其中三个构建体(-3500CAT、-1260CAT和-563CAT)在多种异源细胞系中仅以背景水平表达,这表明sGTHIIβ的5'-侧翼序列包含决定其垂体特异性的信息。鉴定出一个沉默子序列(-95至-35,pSil),它可能在非促性腺细胞或最终成熟和产卵之前的发育阶段的促性腺细胞中抑制sGTHIIβ基因的表达。