Sivashanmugam Perumal, Hall Susan H, Hamil Katherine G, French Frank S, O'Rand Michael G, Richardson Richard T
Department of Cell and Developmental Biology, 206 Taylor Hall, CB# 7090, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, USA.
Gene. 2003 Jul 17;312:125-34. doi: 10.1016/s0378-1119(03)00608-5.
We have recently described a novel gene on human chromosome 20q 12-13.2 called Eppin (Epididymal protease inhibitor) that expresses three mRNAs encoding two isoforms of a cysteine-rich protein containing both Kunitz-type and WAP-type (four disulfide core) consensus sequences (Richardson et al., 2001). To further our studies on Eppin, we have cloned, sequenced and characterized mouse Eppin and report that it lies within a 200 Kb cluster of putative Eppin-like genes on mouse chromosome 2. Analysis of the homologies between the genes in the human and mouse Eppin clusters indicates that the first part of the cluster immediately surrounding Eppin represents a conserved linkage because the order of homologous genes is conserved. Sequencing of reverse transcription polymerase chain reaction (RT-PCR) products confirmed the expression of five of these novel Eppin-like genes in the mouse, which include the mouse homologue of HE-4. These genes are characterized by having either one or both of the Kunitz-type and WAP-type consensus sequences. Additional RT-PCR experiments revealed that expression of some of the Eppin-like genes is restricted to epididymis and testis while others are expressed in several somatic tissues. Northern blot analysis of 22 different mouse tissues identified Eppin transcripts only in the epididymis and testis. Immunostaining of Eppin with anti-recombinant mouse Eppin demonstrated Eppin predominantly on the postacrosomal region of mouse spermatozoa, in Sertoli cells, Leydig cells, and round spermatids in the testis, and in the principal cells of the cauda epididymidis epithelium. Eppin is first expressed by Sertoli cells of 12-day-old mice and subsequently in round spermatids, which is consistent with androgen regulation. Our results demonstrate that mouse chromosome 2 contains a conserved linkage of Eppin-like protease inhibitor genes that are expressed in the epididymis.
我们最近在人类20号染色体12 - 13.2区域发现了一个名为Eppin（附睾蛋白酶抑制剂）的新基因，它表达三种mRNA，编码一种富含半胱氨酸的蛋白质的两种亚型，该蛋白质同时含有Kunitz型和WAP型（四二硫键核心）共有序列（Richardson等人，2001年）。为了进一步研究Eppin，我们克隆、测序并鉴定了小鼠Eppin，报告称它位于小鼠2号染色体上一个200 Kb的假定Eppin样基因簇内。对人和小鼠Eppin基因簇中基因的同源性分析表明，紧邻Eppin的基因簇的第一部分代表保守连锁，因为同源基因的顺序是保守的。逆转录聚合酶链反应（RT-PCR）产物的测序证实了这些新的Eppin样基因中有五个在小鼠中表达，其中包括HE-4的小鼠同源物。这些基因的特征是具有Kunitz型和WAP型共有序列中的一个或两个。额外的RT-PCR实验表明，一些Eppin样基因的表达仅限于附睾和睾丸，而其他基因则在几种体细胞组织中表达。对22种不同小鼠组织的Northern印迹分析仅在附睾和睾丸中鉴定出Eppin转录本。用抗重组小鼠Eppin对Eppin进行免疫染色表明，Eppin主要存在于小鼠精子的顶体后区域、睾丸中的支持细胞、间质细胞和圆形精子细胞以及附睾尾上皮的主细胞中。Eppin首先由12日龄小鼠的支持细胞表达，随后在圆形精子细胞中表达，这与雄激素调节一致。我们的结果表明，小鼠2号染色体包含在附睾中表达的Eppin样蛋白酶抑制剂基因的保守连锁。
