Biomedical subjects
T Zeigler
Publications and source records attributed to T Zeigler.
Flanking and intragenic sequences regulating the expression of the rabbit alpha-globin gene.
Despite their descent from a common ancestral gene and the requirement for coordinated, tissue-specific regulation, the alpha- and beta-globin genes in many mammals are regulated in distinctly different ways. Unlike the beta-globin gene, the rabbit alpha-globin gene is transiently expressed at a high level without an added enhancer in transfected erythroid and non-erythroid cells. By examining a series of alpha/beta fusion genes, we show that internal sequences of the rabbit alpha-globin gene (within the first two exons and introns) are required along with the 5' flank for this enhancer-independent expression. Furthermore, deletion of the introns of the alpha-globin gene, or replacement by introns of the beta-globin gene, results in severely decreased expression of the transfecting genes. Hybrid constructs between segments of the alpha-globin gene and a luciferase gene confirm that internal alpha-globin sequences are needed for high level production of RNA in transfected cells. The flanking and internal sequences implicated in regulation of the rabbit alpha-globin gene coincide with a prominent CpG-rich island and may comprise an extended promoter (including both flanking and intragenic sequences) that is active in transfected cells without an enhancer.
Positive and negative regulatory elements of the rabbit embryonic epsilon-globin gene revealed by an improved multiple alignment program and functional analysis.
The epsilon-globin genes of mammals are expressed in early embryos, but are silenced during fetal and adult erythropoiesis. As a guide to defining the regulatory elements involved in this developmental switch, we have searched the sequences of epsilon-globin genes from different mammals for highly conserved segments. The search was facilitated by the development of a new program, called yama, to generate a multiple alignment of very long sequences using an improved scoring scheme. This allowed us to generate a multiple alignment of sequences from a more divergent group than previously analyzed, as demonstrated here for representatives of four mammalian orders. In parallel experiments, we constructed a series of deletion mutations in the 5' flank of the rabbit epsilon-globin gene and tested their effect on an epsilon-globin-luciferase hybrid reporter gene. These results show that 121 bp of 5' flank, containing CACC, CCAAT and ATA motifs, is sufficient for expression in erythroid K562 cells. Both positive and negative cis-acting control sequences are located between 218 and 394 bp 5' to the cap site, in a region previously proposed to be a silencer. The positive regulatory sequence contains conserved binding sites for the nuclear protein YY1 adjacent to another highly conserved sequence. The negative element contains a conserved sequence followed by a purine-rich segment. This analysis maps the upstream control sequences more precisely and points to a very complex regulatory scheme for this gene.