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We have studied the 5'-flanking sequences required for the transcriptional regulation of human e-globin gene expression. A series of deletion mutants of the human e-globin gene 5'-flanking sequences were constructed and linked to the bacterial chloramphenicol acetyltransferase gene.Expression of these constructs was tested in HeLa cells and the human erythroleukemia K-562 cells. By masuring chloramphenicol acetyltransferase activities and mRNA levels we found that the sequence between -177 and -392 base pairs (bp) relative to the mRNA initiation site exerts a negative effect on E-globin promoter activity. This effect is more pronounced in HeLa cells compared with K-562 cells. To further characterize the negative control region we cloned the DNA sequence between -177 and -392 bp either 5' or 3' of the E-globin promoter and in either orientation. Our data indicate that this negative control region inhibits the e-globin promoter activity in a position-and orientation-independent manner, thus suggesting that it is a silencer. In addition, the silencer also inhibits the expression from the Herpesvirus thymidine kinase promoter. Sequence comparison reveals that there are three short regions within the silencer that share extensive homology with those found in other negative control DNA elements. Our results therefore indicate that an upstream silencer element is present in the E-globin gene and that it may play an important role in the control of e-globin gene expression during development.The human embryonic e-globin gene is a member ofthe A-like globin gene family. This gene is expressed in a tissue-specific and developmental stage-specific manner (1). During early gestation the E-globin gene is expressed in yolk sac islands in the embryo. At -5 weeks of gestation, the site of hematopoiesis shifts to the liver, the embryonic e-globin gene is gradually turned off, and the fetal y-jglobin gene is turned on.This transition is completed well before 10 weeks of gestation. Although there are cellular models to explain the regulation of developmental switching, the molecular mechanisms governing this switching are still not clear.The E-globin gene, like other globin genes, has been shown to contain several promoter elements within the first -100 base pairs (bp) 5' of the RNA initiation site. Two typical higher eukaryotic promoter elements, the TATA box and the CCAAT box, are located at -28 bp and -82 bp, respectively (2). A third conserved sequence, the CCAAC box, which is common to the A-like globin genes (3), is found at -110 bp. Functional analysis by linker-scanning and point mutation revealed that the three conserved elements are necessary for promoter function in the (3-like globin genes (3-7). Deletion analysis showed that the sequences upstream from the CACCC element are not required for constitutive promoter activity (5,6,8). While the known conserved promoter elements seem to play an essential role in promoting nonregulated or constitutive expression of the globin genes, several enhancer elements have bee...

Deinococcus radiodurans and other species of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. Two different DNA damage-sensitive strains generated by chemical mutagenesis were found to be defective in a gene that has extended DNA and protein sequence homology with poL4 of Escherichia coli. Both mutant strains lacked DNA polymerase, as measured in activity gels. Transformation of this gene from wild-type D. radiodurans restored to the mutants both polymerase activity and DNA damage resistance. A technique for targeted insertional mutagenesis in D. radiodurans is presented. This technique was employed to construct a pol mutant isogenic with the wild type (the first example of targeted mutagenesis in this eubacterial family). This insertional mutant lacked DNA polymerase activity and was even more sensitive to DNA damage than the mutants derived by chemical mutagenesis. In the case of ionizing radiation, the survival of the wild type after receiving 1 Mrad was 100%o while survival of the insertional mutant extrapolated to 10-24. These results demonstrate that the gene described here encodes a DNA polymerase and that defects in this pol gene cause a dramatic loss of resistance of D. radiodurans to DNA damage.

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the (Fig. 1A). Strain rec30, which is unusually sensitive to MMC (Fig. 2), was transformed to MMC resistance by genomic DNA from wt (Fig.

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