Gilbert H J, Clarke I N, Gibson R K, Stephenson J R, Tully M
J Bacteriol. 1985 Jan;161(1):314-20. doi: 10.1128/jb.161.1.314-320.1985.
A genomic library of Rhodosporidium toruloides DNA was constructed in bacteriophage lambda 1059. Recombinant phage containing phenylalanine ammonia lyase (PAL) gene sequences were identified by using 32P-labeled cDNA to partially purified PAL mRNA. The PAL gene was subcloned on an 8.5-kilobase PstI DNA restriction fragment into pUC8 to generate the recombinant plasmid pHG2. A restriction map of the PAL gene, together with its flanking regions, was constructed. Northern hybridization analysis of R. toruloides RNA with a restriction fragment encoding part of the PAL gene indicates that PAL mRNA is 2.5 kilobases in length. A single-stranded DNA hybridization probe was constructed and used to quantitate PAL mRNA levels in R. toruloides grown under different physiological conditions. PAL mRNA levels paralleled changes in functional PAL mRNA and antigen. These data are consistent with control of PAL expression being at the level of transcription.
用噬菌体λ1059构建了圆红冬孢酵母DNA的基因组文库。通过使用32P标记的cDNA与部分纯化的苯丙氨酸解氨酶（PAL）mRNA杂交，鉴定出含有PAL基因序列的重组噬菌体。将PAL基因亚克隆到一个8.5千碱基的PstI DNA限制性片段上，插入pUC8中，构建出重组质粒pHG2。构建了PAL基因及其侧翼区域的限制性图谱。用编码部分PAL基因的限制性片段对圆红冬孢酵母RNA进行Northern杂交分析，结果表明PAL mRNA长度为2.5千碱基。构建了单链DNA杂交探针，并用于定量在不同生理条件下生长的圆红冬孢酵母中PAL mRNA的水平。PAL mRNA水平与功能性PAL mRNA和抗原的变化平行。这些数据与PAL表达的调控发生在转录水平一致。
