Shoda Tomoko, Hirata Shuji, Kato Junzo, Hoshi Kazuhiko
Department of Obstetrics and Gynecology, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi, Japan.
J Steroid Biochem Mol Biol. 2002 Oct;82(2-3):201-8. doi: 10.1016/s0960-0760(02)00186-3.
Our recent report has revealed the existence of the progesterone receptor (PR) isoform S, which consists of the novel PR exon S and exons 4-8 of the PR gene in the human testicular cDNA library. More recently, we have cloned the human estrogen receptor alpha (ERalpha) isoform S cDNA from the library. The ERalpha isoform S cDNA also contains the novel ERalpha exon S and exons 4-8 of the ERalpha cDNA. Based on these findings, we assumed that the novel isoform of cDNA like the PR- and ERalpha isoforms might exist in the human ER beta (ERbeta). In order to investigate this possibility, we have screened the human testicular cDNA library using the exons 4-8 corresponding sequence of the human ERbeta cDNA. Consequently, we have cloned a novel isoform of the ERbeta cDNA that consists of a previously unidentified 5'-sequence and the exons 5-8 of the ERbeta gene. We termed this isoform cDNA the "ERbeta isoform M cDNA". The 5'-sequence of the ERbeta isoform M cDNA was confirmed to be derived from a novel exon (termed the "exon M") by analysis of the genomic DNA. Moreover, we have analyzed the molecular size of the ERbeta isoform M encoded by the ERbeta isoform M mRNA by transient expression of the ERbeta isoform M cDNA in the 293T cell. The approximately 28 kDa protein, which was recognized by the anti-rat ERbeta antibody against the carboxyl-terminal region, was synthesized in the cells. Thus, we concluded that the ATG in the exon M could be used as the translation initiation codon. This report revealed for the first time the existence of the ERbeta mRNA isoform that is not caused by the skipping of one or more exons, by the alternative usage of the multiple exon 8s, nor by the alternative utilization of the untranslated 5'-exons located on the upstream region of the exon 1.
我们最近的报告揭示了孕酮受体（PR）同工型S的存在，它由人类睾丸cDNA文库中PR基因的新PR外显子S和外显子4 - 8组成。最近，我们又从该文库中克隆了人类雌激素受体α（ERα）同工型S的cDNA。ERα同工型S的cDNA同样包含新的ERα外显子S和ERα cDNA的外显子4 - 8。基于这些发现，我们推测像PR和ERα同工型一样的新cDNA同工型可能存在于人类雌激素受体β（ERβ）中。为了探究这种可能性，我们使用人类ERβ cDNA的外显子4 - 8对应序列筛选了人类睾丸cDNA文库。结果，我们克隆出了一种新的ERβ cDNA同工型，它由一个此前未鉴定的5'序列和ERβ基因的外显子5 - 8组成。我们将这种同工型cDNA命名为“ERβ同工型M cDNA”。通过对基因组DNA的分析，证实ERβ同工型M cDNA的5'序列源自一个新的外显子（称为“外显子M”）。此外，我们通过在293T细胞中瞬时表达ERβ同工型M cDNA，分析了由ERβ同工型M mRNA编码的ERβ同工型M的分子大小。细胞中合成了一种约28 kDa的蛋白质，它能被针对羧基末端区域的抗大鼠ERβ抗体识别。因此，我们得出结论，外显子M中的ATG可作为翻译起始密码子。本报告首次揭示了ERβ mRNA同工型的存在，它不是由一个或多个外显子的跳跃、多个外显子8的选择性使用，也不是由位于外显子1上游区域的非翻译5'外显子的选择性利用所导致的。
