Differential expression of mouse beta/goat beta c, mouse beta/goat beta F, and mouse beta/goat epsilon II hybrid globin genes in murine erythroleukemia cells.

Abstract

We assembled three hybrid 13-globin (10,12,13,52), and the heat shock-responsive element of the Drosophila hsp-70 gene (9,27 MATERIALS AND METHODSConstruction of fusion genes. We assembled three p-globin fusion genes for this study (Fig. 1) 'n20-kb' 1 jig of HSV-106, enough globin plasmid to give a 10-fold molar excess over HSV-106, and the remainder as highmolecular-weight purified F4-12B2 cell DNA. In the experiments with pUCtk-r, the plasmid was cleaved with ScaI g3C before transformation (Fig. 2); either 1.8 or 9 ,ug of the * pUCtk-e plasmid per plate was used. As in the earlier studies, F4-12B2 DNA was added so that a total of 25 ,ug of DNA was applied to each plate. Regardless of which plasmids were used for transformation, the DNA precipitate was left on the cells for 9 h, when they were fed with nonselective D medium. Twenty hours later, the initial medium was replaced with selective medium (HAT; 15 ,ug of hypoxanthine per ml, 0.2 jiM aminopterin, and 5 jig of thymidine per ml). . DNAs from other lines were dot blotted. Dot blots were or P3F). The mouse prepared by suspending the DNAs in 5 ,ul of 0.1 M NaOH-2 ,B/human 1 construct was that described by Chao et al. (6) and was kindly provided by that laboratory.After our initial round of experiments with the three constructs shown in Fig. 1, we wanted to test the effect of placing the mouse 13/goat Ell gene on the same DNA fragment as the selectable marker gene for thymidine kinase (tk). To accomplish this we first prepared the 1.8-kilobase (kb) PvuII fragment from herpes simplex virus 106 (HSV-106) (24) which contains the entire herpesvirus tk gene; this was cloned by blunt-end ligation into the unique SspI site of pUC18. The EcoRI fragment containing the mouse 1/goat Ell gene was then cloned into the BamHI site in the polylinker of the pUCtk plasmid by blunt-end ligation, creating plasmid pUCtk-r. This construct is diagrammed in Fig. 2.Introduction of globin genes into MEL cells. The F4-12B2