Bradley C C, Blakely R D
Medical Scientist Training Program, Emory University School of Medicine, Atlanta, Georgia, U.S.A.
J Neurochem. 1997 Oct;69(4):1356-67. doi: 10.1046/j.1471-4159.1997.69041356.x.
To explore the structural basis for regulation of human serotonin transporter (hSERT) gene expression, we used primer extension and 5' rapid amplification of cDNA ends (5'RACE) techniques to estimate levels of and identify 5'-noncoding elements of hSERT mRNAs and genomic cloning to place these elements within the overall map of the hSERT gene. Primer extension on JAR cell mRNA suggested the presence of significant hSERT mRNA sequence upstream of the 5' end of our cloned hSERT cDNA. Using 5'RACE and reverse transcription-PCR (RT-PCR) methodologies, we cloned these sequences from brain and placenta and found this material to be composed of alternatively spliced exons using a previously reported noncoding exon (1A) and a novel 97-bp noncoding exon (1B). RT-PCR of JAR cell mRNA blotted with exon-specific oligonucleotides revealed both exons 1A and 1B to be regulated in a cholera toxin-dependent manner. To clarify the structure of the hSERT gene including exon 1B, we isolated and characterized genomic hSERT clones from Lambda Fix II and P1 artificial chromosome libraries. In agreement with previous findings, a single hSERT gene was identified that accounts for hybridizing bands on genomic Southern blots and was found to utilize 13 exons to encode the transporter's coding sequences along with the two noncoding 5' exons. Exon 1B was identified approximately 14 kb downstream of exon 1A in the hSERT gene and 737 bp upstream of exon 2, where the initiation site for translation is located. Exon 1B is surrounded by several elements potentially suitable for regulating serotonin transporter gene expression in vivo, including consensus sites for transcription factors AP-1, AP-2, CREB/ATF, and NF-kappaB. These data reveal additional complexity in hSERT gene structure and expression that may be relevant to regulated and compromised transporter expression in vivo.
为了探究人类血清素转运体（hSERT）基因表达调控的结构基础，我们运用引物延伸和5' cDNA末端快速扩增（5'RACE）技术来估算hSERT mRNA的水平并鉴定其5'非编码元件，同时采用基因组克隆技术将这些元件定位到hSERT基因的整体图谱中。对JAR细胞mRNA进行引物延伸分析表明，在我们克隆的hSERT cDNA的5'端上游存在大量hSERT mRNA序列。利用5'RACE和逆转录-聚合酶链反应（RT-PCR）方法，我们从脑和胎盘中克隆了这些序列，发现这些物质由使用先前报道的非编码外显子（1A）和一个新的97 bp非编码外显子（1B）进行可变剪接的外显子组成。用外显子特异性寡核苷酸印迹的JAR细胞mRNA的RT-PCR显示，外显子1A和1B均以霍乱毒素依赖的方式受到调控。为了阐明包括外显子1B在内的hSERT基因的结构，我们从Lambda Fix II和P1人工染色体文库中分离并鉴定了基因组hSERT克隆。与先前的研究结果一致，我们鉴定出一个单一的hSERT基因，它解释了基因组Southern印迹上的杂交条带，并发现该基因利用13个外显子来编码转运体的编码序列以及两个5'非编码外显子。在hSERT基因中，外显子1B位于外显子1A下游约14 kb处，外显子2上游737 bp处，而外显子2是翻译起始位点所在位置。外显子1B周围有几个可能适合在体内调控血清素转运体基因表达的元件，包括转录因子AP-1、AP-2、CREB/ATF和NF-κB的共有位点。这些数据揭示了hSERT基因结构和表达中额外的复杂性，这可能与体内转运体表达的调控和受损有关。
