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D M Bodine

Publications and source records attributed to D M Bodine.

78 records · Page 5Linked to original sources
PubMed1988

Expression of the human beta-globin gene following retroviral-mediated transfer into multipotential hematopoietic progenitors of mice.

Efficient transfer of the beta-globin gene into primitive hematopoietic progenitors was achieved with consistent and significant expression in the progeny of those cells. Retroviral vectors containing the intact genomic human beta-globin gene and the neomycin (G418)-resistance (neoR) gene were constructed. These gave titers of 10(6) or more neoR colony-forming units/ml when packaged in psi 2 cells. Mouse bone marrow cells were infected by coculture with producer cells and injected into lethally irradiated animals. Several parameters were varied to enhance infection frequency of colony-forming units, spleen (CFU-S); overall 41% of 116 foci studied contained an intact proviral genome. The human beta-globin gene was expressed in 31 of 35 CFU-S-derived spleen colonies that contained the intact vector genome at levels ranging from 1% to 5% of that of the mouse beta-globin genes. Infected bone marrow cells were also injected into genetically anemic W/Wv recipients without prior irradiation. Human beta-globin chains were detected in circulating erythrocytes by immunofluorescent staining with a specific monoclonal antibody. All animals injected with donor cells that had been cultured in G418 (1 mg/ml) for 48 hr after retroviral infection had circulating erythrocytes containing human beta-globin chains between 3 and 8 weeks after transplantation.

Animals↗
PubMed1987

An enhancer element lies 3' to the human A gamma globin gene.

We have surveyed 22 kb of DNA from the region surrounding the human fetal (gamma) globin genes and have identified one fragment that meets all of the criteria for a non-tissue specific enhancer element. The enhancer-containing fragment starts approximately 400 bp 3' to the polyadenylation signal of the A gamma gene and is less than 750 bp in length. Addition of this fragment to plasmids containing a 'gamma-CAT' hybrid gene [consisting of the gamma globin gene promoter fused to the chloramphenicol acetyl transferase (CAT) gene] increases CAT expression 6-23-fold in K562 erythroleukemia cells, depending upon the method of transfection. The increase in expression is essentially independent of the orientation or position of the fragment with respect to the gamma-CAT hybrid gene. The 3' gamma enhancer activates heterologous promoters in erythroleukemia cells, and is also active in non-erythroid cell lines. The enhancer acts by increasing the number of transcripts initiated from the normal gamma globin gene transcription initiation site. The enhancer region contains two DNase I hypersensitive sites in erythroleukemia cells but none in nonerythroid human leukemia cell lines. The 3' gamma globin gene enhancer contains a unique element that is similar to sequences found in an enhancer 3' to the chicken beta globin gene, suggesting that this conserved element may have a role in enhancer function.

Base Sequence↗
PubMed1985

Remarkable homology among the internal repeats of erythroid and nonerythroid spectrin.

A cDNA clone for nonerythroid alpha-spectrin was identified by direct immunological screening of a chicken smooth muscle cDNA library. A library prepared in the expression plasmids pUC8 and pUC9 was screened with an antiserum specific for chicken alpha-spectrin. Blots of poly(A)+ RNA from various tissues of chicken and mouse show that the cDNA hybridizes to an 8-kilobase mRNA. The cDNA hybridizes to a single-copy sequence on Southern blots of chicken genomic DNA. The complete nucleic acid sequence of the clone has a single 1419-base open reading frame. The derived amino acid sequence is organized into two partial and three complete 106-amino-acid repeats that show homology to the repeats described for human erythroid alpha- and beta-spectrin. Immunological and biochemical data indicate that chicken nonerythroid and human erythroid alpha-spectrin are two of the more widely diverged members of the spectrin family of proteins. In this respect, the degree of homology found between them was unexpected. Our data suggest a common evolutionary origin for these two alpha-spectrins and allow some predictions concerning spectrin gene structure.

Amino Acid Sequence↗
PubMed1984

Spectrin deficient inherited hemolytic anemias in the mouse: characterization by spectrin synthesis and mRNA activity in reticulocytes.

We have investigated spectrin synthesis and mRNA activity in mice homozygous and heterozygous for six mutations occurring at three distinct loci (nb, ja, sph). When homozygous, these mutations cause severe hemolytic anemias that are characterized by specific spectrin deficiencies. Our results indicate that the primary effect of the nb mutation is a deficiency of another erythrocyte membrane skeletal protein, ankyrin. The severe deficiency of spectrin in the red blood cells of ja/ja mice is the result of a beta spectrin defect. Analysis of spectrin synthesis in mice homozygous and heterozygous for several alleles of sph indicates that the sph locus is the structural gene locus for alpha spectrin. We have mapped the sph locus to mouse Chromosome 1.

Anemia, Hemolytic↗
PubMedDate not supplied

An erythroid-specific chromatin opening element reorganizes beta-globin promoter chromatin structure and augments gene expression.

In erythroid tissues the chromatin structure of the beta-globin gene locus is extensively remodeled. Changes include the formation of DNase I hypersensitive sites (HSs) over the promoters of actively expressed genes. To test the hypothesis that such "opening" of promoter chromatin structure is important for beta-globin gene expression, we placed a 101-bp erythroid-specific hypersensitive-site forming element (HSFE) from the core of LCR HS4 immediately upstream of a minimal beta-globin gene promoter. We then studied the effects of this element alone and in combination with other cis-acting elements on globin gene chromatin structure and gene expression in MEL cells and transgenic mice. Single or tandem HSFEs increased the size of the portion of the promoter accessible to DNase digestion, increased the proportion of promoters in an accessible conformation, and increased gene expression approximately 5-fold. These were equivalent to expression levels attained using a 2.8-kb microLCR construct. Inclusion of the LCR HS2 enhancer did not increase expression further. In transgenic mouse fetal liver cells the HSFE increased average expression 2.5-fold compared to the minimal promoter alone. These results indicate that a small cis-acting element is capable of remodeling local beta-globin promoter chromatin structure and producing expression similar to that seen with a microLCR construct.

Animals↗