Publication in refereed journal
香港中文大学研究人员 ( 现职)
| 崔耀隆教授 (化学病理学系) |
全文
| 数位物件识别号 (DOI) http://dx.doi.org/10.1006/jmbi.1995.0318 |
引用次数
Web of Sciencehttp://aims.cuhk.edu.hk/converis/portal/Publication/13WOS source URL
Scopushttp://aims.cuhk.edu.hk/converis/portal/Publication/13Scopus source URL
其它资讯
摘要The affinity maturation of antibodies is driven by somatic hypermutation which is localized to specific segments of the coding genes. The information available on this process derives from studies in vivo. With the intention of developing new approaches, we have constructed a fusion gene between a kappa chain and a selectable neomycin resistance gene, neor. The neor gene, which includes the SV40 small t intron and polyadenylation site, but not the upstream elements nor its first 12 amino acids, is an in-frame substitution of the FR2-CDR3 fragment of a rearranged VκOx1-Jκ5 gene. Expression of neor activity is therefore dependent on the upstream immunoglobulin sequence. A stop codon was placed in the CDR1 region so that only mutants survive treatment with geneticin sulphate (G418). The effectiveness of the system was tested by transfecting the NS0 myeloma cell line and isolating spontaneous mutants. Neomycin-resistant clones arose at an estimated rate of 1 × 10-8/cell division, and over 90% were authentic structural mutants. Unlike the somatic hypermutations, the majority arose by in-frame deletions including the stop codon, although up to 30% involved a point mutation. The reporter gene was then modified by substituting all the sequences downstream of the Jκ5 with others known to be required for full hypermutation in vivo. Different cell lines were transfected and G418-resistant clones analyzed. No significant increase in the rate of reversion or in the generation of point mutations versus deletions was detected, even using conditioned culture medium. In the presence of azacytidine however, a mutant involving multiple events (single base addition and deletion plus two point mutations) was detected. The reporter gene system therefore seems suitable to test culture conditions and modifications of the host cells aimed at the derivation of an in vitro assay of somatic hypermutation.
着者Chui Y.L., Lozano F., Jarvis J.M., Pannell R., Milstein C.
期刊名称JOURNAL OF MOLECULAR BIOLOGY
出版年份1995
月份12
日期1
卷号249
期次3
出版社Academic Press
出版地United States
页次555 - 563
国际标準期刊号0022-2836
电子国际标準期刊号1089-8638
语言英式英语
关键词Somatic mutations, Spontaneous mutations, Stop-codon revertants, Tissue culture mutants
