PHYSICAL MAPPING OF THE HUMAN T-CELL RECEPTOR-BETA GENE-COMPLEX, USING YEAST ARTIFICIAL CHROMOSOMES

Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human T-cell beta chain gene complex (TCRB). Varia ble region genes (BV) for the 25 known subfamilies were used as probes to screen the ICRF AM4x YAC library. Of the five positive YACs identi fied, one YAC designated B3, 820 kilobase pairs (kbp) in size, scored positive for all 25 TCRBV subfamilies plus the constant region genes ( BC) when analyzed by pulse field gel electrophoresis. Restriction enzy me mapping of B3 located TCRBV and TCRBC gene regions to 4 Sfi I fragm ents of 280 110, 90, and 125 kbp and was in accordance with published data. In addition comparison of hybridization results of Sfi I-restric ted B3 and genomic DNA from the parental cell line GM1416B revealed id entical banding patterns. The data thus showed YAC B3 encoded a comple te and unrearranged TCRB gene locus of some 600-620 kbp. The map was f urther resolved by locating restriction sites for Sal I and Bss HII on B3, giving more precise localization of the individual TCRBV gene fam ilies. Fluorescent in situ hybridization of B3 to spreads of human met aphase chromosomes localized B3 to 7q35. However, two additional signa ls were obtained; one attributable to the TCRBV orphon cluster on 9p21 , the second to the long arm of chromosome 2. Polymerase chain reactio n amplification of a chromosome 2 somatic cell hybrid, using primers f or all 25 TCRBV gene families, revealed that the signal was not attrib utable to a second orphon cluster. It is suggested that B3 is a chimer ic YAC with an intact TCRB locus flanked by chromosome 2 sequences.