A new T-lymphocyte cloning assay for detection of in vivo mutations in the human hypoxanthine-guanine phosphoribosyltransferase gene

✍ Scribed by Kazutsugu Uematsu; Jun Yasuda; Teruaki Sekine; Kokichi Sugano; Shoji Kudoh; Takao Sekiya

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 148 KB
• Volume: 30
• Category: Article
• ISSN: 0893-6692
• DOI: 10.1002/(sici)1098-2280(1997)30:1<31::aid-em5>3.0.co;2-f

✦ Synopsis

The X-linked hypoxanthine-guanine phosphoribosyl-respectively. These data were similar to the reported transferase (hprt) gene is a target of analyses of in values. The mean MFs in the nine colon cancer pavivo mutation frequencies in circulating T-lympho-tients (10.6 { 7.3 1 10 06 ) were not significantly cytes. We established a novel, accessory cell-free different from those of the 12 cancer-free individuals cloning method of T-lymphocytes with a hprt muta-(11.6 { 9.4 1 10 06 ). The correlation between mution by a combined use of recombinant interleukin-tation frequencies and age of the individuals was 2, conditioned medium from activating T-lympho-significant regardless of the presence or absence cytes and culture plates coated with anti-CD3 mono-of cancers. The single-strand conformation polymorclonal antibody. Using the method, we examined phism analyses of nested RT-PCR products of hprt mutation frequencies of the hprt gene in T-lympho-mRNA were done in 33 mutant clones from five cytes from six healthy individuals, nine patients with members of a family of which MF values were high. colon cancer including two patients from different All the analyzed mutant clones show a genetic aberfamilies with hereditary nonpolyposis colon cancer ration in the coding region of the hprt gene. At least and six cancer-free relatives of the patients. In six 28 of 33 mutants were independent. Our method healthy individuals, the mean cloning efficiency and provides a new versatile tool for in vivo analysis for mutation frequency (MF) of the hprt gene in T-lym-mutations of the hprt gene. Environ. Mol. Mutagen. phocytes were 0.51 { 0.28 and 9.4 { 7.5 1 10 06 , 30: 31-39, 1997 ᭧ 1997 Wiley-Liss, Inc.