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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021313-11
Application #
3270391
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1974-10-01
Project End
1987-11-30
Budget Start
1984-12-01
Budget End
1985-11-30
Support Year
11
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Mears, M L; Hutchison 3rd, C A (2001) The evolution of modern lineages of mouse L1 elements. J Mol Evol 52:51-62
Montague, M G; Hutchison 3rd, C A (2000) Gene content phylogeny of herpesviruses. Proc Natl Acad Sci U S A 97:5334-9
Wrobel, J A; Conrad, M J; Bloedon, E et al. (2000) Analysis of HIV type 1 reverse transcriptase: comparing sequences of viral isolates with mutational data. AIDS Res Hum Retroviruses 16:2049-54
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Wrobel, J A; Chao, S F; Conrad, M J et al. (1998) A genetic approach for identifying critical residues in the fingers and palm subdomains of HIV-1 reverse transcriptase. Proc Natl Acad Sci U S A 95:638-45
Tollefsbol, T O; Hutchison 3rd, C A (1998) Analysis in Escherichia coli of the effects of in vivo CpG methylation catalyzed by the cloned murine maintenance methyltransferase. Biochem Biophys Res Commun 245:670-8
Tollefsbol, T O; Hutchison 3rd, C A (1997) Control of methylation spreading in synthetic DNA sequences by the murine DNA methyltransferase. J Mol Biol 269:494-504
Davies, C J; Hutchison 3rd, C A (1995) Insertion site specificity of the transposon Tn3. Nucleic Acids Res 23:507-14
Peterson, S N; Bailey, C C; Jensen, J S et al. (1995) Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation. Proc Natl Acad Sci U S A 92:11829-33
Tollefsbol, T O; Hutchison 3rd, C A (1995) Mammalian DNA (cytosine-5-)-methyltransferase expressed in Escherichia coli, purified and characterized. J Biol Chem 270:18543-50

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