Yamauchi M, Yamauchi N, Phear G, Spurr N K, Martinsson T, Weith A, Meuth M
Cell Mutation Laboratory, Imperial Cancer Research Fund, Clare Hall Laboratories, Potters Bar, Hertfordshire, United Kingdom.
Genomics. 1991 Dec;11(4):1088-96. doi: 10.1016/0888-7543(91)90036-e.
To elucidate the organization of the human genomic sequences encoding CTP synthetase (CTPS), fragments homologous to the cDNA were isolated from genomic lambda libraries. The fragments cloned were overlapping and cover over 40 kb. Cotransfection of the DNAs into CTPS-deficient, cytidine-requiring CHO mutants can transform them to cytidine-independent growth, indicating that the complete structural gene has been isolated. Direct sequencing and enzymatic amplification of the cloned genomic fragments revealed that the coding sequences are distributed to 19 exons covering about 35 kb. Multiple transcriptional start sites were detected by primer extension in a G + C-rich 5' flanking sequence that is separated from the translational start by an approximately 3-kb intron. A panel of human-rodent somatic cell hybrids and the CTPS cDNA were used to assign the structural gene to the short arm of human chromosome 1. This assignment was further refined through the use of somatic cell hybrids bearing fragments of the short arm of the chromosome, allowing localization to 1p36.11-p31, a region notable for its disruption in many types of tumors.
为了阐明编码CTP合成酶（CTPS）的人类基因组序列的组织情况，从基因组λ文库中分离出与cDNA同源的片段。克隆的片段相互重叠，覆盖超过40 kb。将这些DNA共转染到缺乏CTPS、需要胞苷的CHO突变体中，可使其转化为不依赖胞苷的生长，这表明已分离出完整的结构基因。对克隆的基因组片段进行直接测序和酶促扩增，结果显示编码序列分布在19个外显子中，覆盖约35 kb。通过引物延伸在富含G + C的5'侧翼序列中检测到多个转录起始位点，该序列与翻译起始位点之间被一个约3 kb的内含子隔开。利用一组人-啮齿动物体细胞杂种和CTPS cDNA将结构基因定位到人类染色体1的短臂上。通过使用携带该染色体短臂片段的体细胞杂种进一步精确了该定位，将其定位到1p36.11 - p31，该区域在许多类型的肿瘤中均有破坏，因而引人注目。
