Kim K S, Wessel T C, Stone D M, Carver C H, Joh T H, Park D H
Laboratory of Molecular Neurobiology, Cornell University Medical College, Burke Rehabilitation Center, White Plains, NY 10605.
Brain Res Mol Brain Res. 1991 Mar;9(4):277-83. doi: 10.1016/0169-328x(91)90073-7.
Tryptophan hydroxylase (TPH) from central serotonergic neurons in the dorsal raphe nucleus (DRN) and that from the endocrine pineal gland (PG) have been shown to exhibit difference biochemical characteristics. We further report here that the isoelectric point determined by chromatofocusing differs between TPH from the rat brainstem and PG. In addition, the levels of TPH mRNA are much greater in the PG than the DRN despite a higher enzymatic activity in the DRN. These data raise the question as to whether different forms of TPH may exist in the DRN and the PG. To address this question, we amplified TPH cDNAs by the polymerase chain reaction (PCR) using poly(A)+ RNA purified from both tissues. Several combinations of oligonucleotide primers encompassing different regions of the published coding sequence of rat pineal TPH were employed for this purpose. Subsequent analysis by gel electrophoresis and Southern blotting of PCR products indicated that DNA fragments of identical length were amplified from both sources. Furthermore, the nucleotide sequences of three independent subclones containing the putative full-length coding region of DRN TPH were determined and found to be identical to that of PG. In situ hybridization using the amplified cDNA as a probe demonstrated specific labeling within the DRN of the rat brain. These data support the hypothesis that tissue-specific differences in TPH characteristics result from differential post-translational events and clearly indicate that a TPH mRNA transcript identical in coding sequence to the PG form is expressed in the DRN.
中缝背核（DRN）中中枢5-羟色胺能神经元的色氨酸羟化酶（TPH）与内分泌松果体（PG）中的色氨酸羟化酶已被证明具有不同的生化特性。我们在此进一步报告，通过色谱聚焦法测定的大鼠脑干和PG中TPH的等电点有所不同。此外，尽管DRN中的酶活性较高，但PG中TPH mRNA的水平比DRN中的高得多。这些数据提出了一个问题，即DRN和PG中是否可能存在不同形式的TPH。为了解决这个问题，我们使用从这两种组织中纯化的聚腺苷酸加尾RNA（poly(A)+ RNA），通过聚合酶链反应（PCR）扩增TPH cDNA。为此，使用了几种包含已发表的大鼠松果体TPH编码序列不同区域的寡核苷酸引物组合。随后通过凝胶电泳和PCR产物的Southern印迹分析表明，从两个来源扩增出了长度相同的DNA片段。此外，测定了三个独立亚克隆的核苷酸序列，这些亚克隆包含DRN TPH假定的全长编码区，发现与PG的相同。使用扩增的cDNA作为探针进行原位杂交，在大鼠脑的DRN内显示出特异性标记。这些数据支持了这样的假设，即TPH特性的组织特异性差异是由翻译后事件的差异导致的，并且清楚地表明在DRN中表达了一种编码序列与PG形式相同的TPH mRNA转录本。
