Restriction digests of the clone give the following sizes (kb): AccI--6.6; AvaI--6.6; EcoRI--2.5, 1.7, 1.4, 1.3. The insert contains the following restriction sites (nt position according to the published sequence): XbaI--258, 1924; SacI--733; BamHI--1273; EcoRI--1770, 3040; AvaI--3425. The 3' HindIII site and the 5' AccI site were lost in cloning. The DNA-binding domain of the protein extends from aa 603 to aa 668 (encoded from nt 2029 to 2226). The ligand-binding domain of the protein extends from aa 734 to aa 984. Contains the AccI/HindIII (nt 188 to nt 3562) fragment from lambdahK10, which includes the complete coding sequence (nt 217 to nt 3171). Expresses functional receptor when transfected into COS-1 cells. The 1.27 kb EcoRI fragment (nt 1770 to nt 3040) has been used to detect mRNA in several rat tissues. The EcoRI fragment of lambdahk10 (nt 1 to nt 3750) was inserted into pGEM-4, excised with HindIII (polylinker to nt 3562), and ligated to the HindIII/HpaI fragment of pRSVcat. The AccI fragment (about 200 bp) at the 5' end was removed to improve translation, and the PvuII/HindIII fragment from pSV2cat containing the SV40 ori was linkered with NdeI and inserted at the NdeI site. |
