We propose to explore the organization of the murie beta-globin complex locus (Hbb) in great detail. We will complete the DNA sequencing of the beta homologous genes, and begin sequencing the intergenic regions. This is part of a long term effort to generate the complete sequence of this 65 kb complex locus. We are particularly interested in detailed structural analysis of the repetitive sequence elements within the complex, and in insertion/deletion differences between the (HBB)d and (Hbb)s haplotypes. We will look extensively for the embryonic Hbb-z gene, which has so far evaded detection in our clones. We will complete our mapping of a new (Hbb) complex, from the mouse Mus pahari. We also plan to study expression of the sequences of the Hbb complex. We will determine whether transcripts of the Betah phi and/or Betah1 structures, which we have observed in MEL cells, are translated to produce a beta-like globin. We will survey the complex for non-globin transcripts (both Pol II and Pol III) in vitro, and in vivo. We will isolate clones of non-globin genes which are expressed during red blood cell differentation, in order to investigate whether these share any sequence features with the genes of the (Hbb) complex. We will explore the use of DNA microinjection into fertilized eggs, as an assay system for sequences involved in gene regulation and switching. We will inject adult and non-adult globin genes, along with enough flanking sequences to provide context. We will also investigate the regulation of hybrid globin genes constructed in vitro. We will also inject a strong eukaryotic promoter (Friend virus LTR) linked to a unique assay sequence (from phage phi X174). We will investigate the surrounding sequencing sequences, at the site of integration, in mice which show tissue specific transcription of the assay sequence.
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