Dahl H H, Mercer J F
J Biol Chem. 1986 Mar 25;261(9):4148-53.
Screening of a rat liver cDNA expression library constructed in the vector lambda gt11 with an affinity purified antiserum to rat phenylalanine hydroxylase has resulted in the isolation of two clones which contain the complete coding region (1362 base pairs) of phenylalanine hydroxylase and the entire 3'-untranslated region (562 base pairs). From the nucleotide sequence we deduced the amino acid sequence of the enzyme. The molecular weight is 51,632 (452 amino acids). The rat enzyme is highly homologous to human phenylalanine hydroxylase. The two proteins differ in only 36 amino acids (92% homology), many of which are conservative changes. A dot matrix computer program was used to analyze regions of homology with the amino acid sequence of rat tyrosine hydroxylase. Considerable homology was detected from amino acid 140 in the rat enzyme to the C terminus, but little or no homology was apparent in the N-terminal region. The cDNA clone was used to determine the levels of phenylalanine hydroxylase mRNA in rat tissues using RNA blot hybridization. Two mRNA species were detected, with approximate lengths of 2,000 and 2,400 nucleotides, which appear to derive from use of alternate polyadenylation signals. No difference in mRNA size was found in rats which have different phenylalanine hydroxylase alleles. The kidney was found to contain about 10% of the mRNA found in the liver, and no phenylalanine hydroxylase mRNA was detected in rat brain. Reuber H4 hepatoma cells were also analyzed for phenylalanine hydroxylase mRNA. The parental cells contained mRNA species of the same sizes as in rat liver. Incubation in 10(-6) M hydrocortisone for 24 h resulted in an 18-fold increase in the mRNA level. Mutant hepatoma cells which express very little phenylalanine hydroxylase contained less than 5% of the parental mRNA, but the gene still responded to hydrocortisone.
用亲和纯化的抗大鼠苯丙氨酸羟化酶血清筛选以λgt11为载体构建的大鼠肝脏cDNA表达文库，已分离出两个克隆，它们包含苯丙氨酸羟化酶的完整编码区（1362个碱基对）和整个3'非翻译区（562个碱基对）。从核苷酸序列我们推导了该酶的氨基酸序列。分子量为51,632（452个氨基酸）。大鼠酶与人苯丙氨酸羟化酶高度同源。这两种蛋白质仅在36个氨基酸上不同（92%同源性），其中许多是保守性变化。用点阵计算机程序分析与大鼠酪氨酸羟化酶氨基酸序列的同源区域。在大鼠酶的第140位氨基酸到C末端检测到相当高的同源性，但在N末端区域几乎没有同源性。cDNA克隆用于通过RNA印迹杂交测定大鼠组织中苯丙氨酸羟化酶mRNA的水平。检测到两种mRNA种类，长度约为2000和2400个核苷酸，它们似乎来自交替使用的聚腺苷酸化信号。在具有不同苯丙氨酸羟化酶等位基因的大鼠中未发现mRNA大小的差异。发现肾脏中含有的mRNA约为肝脏中的10%，在大鼠脑中未检测到苯丙氨酸羟化酶mRNA。还对鲁伯H4肝癌细胞进行了苯丙氨酸羟化酶mRNA分析。亲本细胞含有与大鼠肝脏中大小相同的mRNA种类。在10^(-6)M氢化可的松中孵育24小时导致mRNA水平增加18倍。表达很少苯丙氨酸羟化酶的突变肝癌细胞所含的mRNA不到亲本细胞的5%，但该基因仍对氢化可的松有反应。
