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Locus control region (LCR) is known to occur 5'-upstream of the globin gene clusters in humans and a number of other animals. It comprises four DNase I hypersensitive sites, HS 1-4, and has been considered to play a key role in regulating the globin gene expression in tissue- and developmental stage-specific manners. The occurrence of LCR in the rat genome, however, has not been documented so far. In the present study, the author intended to identify and analyze the rat beta-LCR HS 1 and HS 2, in order to further facilitate studies on the regulatory mechanism involved in globin gene expression. The results obtained in this study are summerized as follows: 1. A DNA region of about 700 bp on the rat genome was amplified by polymerase chain reaction (PCR) using synthetic primers derived from portions of the mouse beta-LCR HS 2. The nucleotide sequence of the PCR product (R 700) shows 67% and 83% homologies with those of the human and mouse HS 2, respectively, indicating that R 700 represents beta-LCR HS 2 of rats. 2. In order to locate beta-LCR HS 2 on the rat genome, a 7 kb DNA fragment (R 7,000) harboring a region between beta-LCR HS 2 and the epsilon 1-globin gene was obtained by PCR. Restriction endonuclease mapping of R 7,000 revealed that the rat beta-LCR HS 2 is located 6.0 kb 5'-upstream relative to the cap site of the epsilon 1-globin gene. 3. The rat beta-LCR HS 1 was then located 4.2 kb 5'-upstream of the epsilon 1-globin gene by Southern blot hybridization of R 7,000 using a human HS 1 probe. Nucleotide sequencing revealed that the rat HS 1 has 83% homology to the mouse HS 1. 4. Comparisons of the structures of the rat beta-LCR HS 1 and HS 2 with those of other animal species indicate that several motifs and consensus sequences for binding of transcription factors, such as NF-E 2/AP-1 and GATA-1, are well conserved during evolutional periods, indicating an indispensable role of LCR in globin gene expression.

Identification and characterization of the rat .BETA.-globin locus control region(LCR) site 1 and site 2.