We have constructed a "minilocus" that contains the 5' and 3' flanking regions of the human beta-globin locus and the beta-globin gene. These regions are characterized by erythroid-specific DNAase I-superhypersensitive sites and are normally located approximately 50 kb 5' and 20 kb 3' of the beta-globin gene. This minilocus is expressed tissue-specifically in transgenic mice at a level directly related to its copy number yet independent of its position of integration in the genome. Moreover, the expression per gene copy is the same in each mouse and as high as that of the endogenous mouse beta-globin gene. These results indicate that the DNA regions flanking the human beta-globin locus contain dominant regulatory sequences that specify position-independent expression and normally activate the complete human multigene beta-globin locus.

We have introduced a human beta-globin minilocus, containing the recently described dominant control region (DCR), the beta-globin or Thy-1 gene, and a thymidine kinase (tk)-neoR gene into erythroid and non-erythroid cells. Analysis of the transcription levels of the genes shows that the DCR directs high levels of human beta-globin, Thy-1 and tk-neo expression independent of integration sites in an erythroid-specific manner. The presence of the DNAasel hypersensitive sites at the 5' end of the locus is required for this effect on the homologous and heterologous gene. An analysis of the DCR chromatin in transfected mouse erythroleukemic cells suggests that the formation of the hypersensitive sites in this region precedes beta-globin gene expression.

The fission yeast cdc21 protein belongs to the MCM family, implicated in the once per cell cycle regulation of chromosome replication. In budding yeast, proteins in this family are eliminated from the nucleus during S phase, which has led to the suggestion that they may serve to distinguish unreplicated from replicated DNA, as in the licensing factor model. We show here that, in contrast to the situation in budding yeast, cdc21 remains in the nucleus after S phase, as is found for related proteins in mammalian cells. We suggest that regulation of nuclear import of these proteins may not be an essential aspect of their function in chromosome replication. To determine the function of cdc21+, we have analysed the phenotype of a gene deletion. cdc21+ is required for entry into S phase and, unexpectedly, a proportion of cells depleted of the gene product are able to enter mitosis in the absence of DNA replication. These results are consistent with the view that individual proteins in the MCM family are required for all initiation events, and defective initiation may impair the coordination between mitosis and S phase.

Ultraviolet (UV) light induces a variety of lesions in DNA of which the pyrimidine dimer represents the major species. Pyrimidine dimers exist as both a cyclobutane type and a 6-4' (pyrimidine-2'-one) photoproduct. We have purified a protein of M(r) approximately 125,000 from HeLa cell nuclei which binds efficiently to double-stranded DNA irradiated with UV light but not to undamaged DNA. This protein was designated UVBP1 (UV damage binding protein 1). UVBP1 did not recognise DNA damaged by cisplatin. Using oligonucleotides with a single dipyrimidine site for induction of UV photoproducts, binding of UVBP1 to a TC-containing substrate was shown to be more efficient than to substrates containing a TT, a CT or a CC pair. This binding specificity implies selective recognition of the 6-4' photoproduct. Further evidence for this was provided by the finding that hot alkali treatment of the substrate (which selectively hydrolyses 6-4' photoproducts) abrogated binding of UVBP1, whereas incubation with DNA photolyase to remove cyclobutane dimers did not. No detectable DNA helicase, ATPase or exonuclease activity was associated with the purified protein. We suggest that UVBP1 may be involved in the lesion recognition step of DNA excision repair and could contribute to the preferential repair of 6-4' photoproducts from the DNA of UV-irradiated mammalian cells.