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For the treatment of beta-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the beta6 position of the mouse beta-major globin gene (beta(6I)) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked beta-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant beta-major and beta-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the beta(6I) mRNA was indistinguishable from that of the wild-type counterpart in bone marrow cells. Similarly, the output from the beta-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult beta-globin genes. (Blood. 2001;98:65-73)

The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined. The dimerization constant for cro has been measured as a prelude to these binding studies. Here, the energetics of cro binding to O(R) are evaluated using quantitative DNaseI footprint titration techniques. Binding data for wild-type and modified O(R) site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O(R) sites. Binding of cro dimers is strongest to O(R)3, then O(R)1 and lastly, O(R)2. Adjacently bound repressors exhibit positive cooperativity ranging from -0.6 to -1.0 kcal/mol. Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation. This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI-cro cooperativity., (Copyright 2000 Academic Press.)

The software program, HINT (Hydropathic INTeractions), which characterizes non-polar-non-polar, polar-polar, and non-polar-polar interactions, has been used to examine subunit interface associations involved in the hemoglobin allosteric transition at a residue and atomic level. HINT differs from many other computational programs in that it is based not on a statistical method or a force-field but employs parameters experimentally determined from solvent transfer experiments. The main focus of this study is to compare HINT scores that are based upon experimentally and thermodynamically derived measurements with experimentally determined thermodynamic results. The HINT analysis yields a good first-order approximation of experimentally measured energies for these interactions as determined by free energies of dimer-tetramer assembly for mutant hemoglobins. The results provide a framework for understanding subunit stabilities based upon individual atom interactions and repulsions. HINT, in agreement with previous analyses, indicates that: (1) the alpha1beta1 and alpha2beta2 subunit contacts are stabilized via several polar and many hydrophobic interactions with few repulsive contact areas in both the T (deoxyhemoglobin) and R (oxyhemoglobin) structures; (2) the alpha1alpha2 subunit contacts are primarily stabilized by polar salt bridge linkages in both T and R states; and (3) the alpha1beta2 and alpha2beta1 contacts have both strong positive and negative interactions in both T and R states with few hydrophobic interactions. The HINT scoring methodology provides a quantitative characterization of the major role of the alpha1beta2 and alpha2beta1 interfaces in the T-->R quaternary transition. HINT also confirms the stronger hydrogen bond formation in mutant Hb Rothschild (Trp 37beta-->Arg) with Asp94alpha1 that gives rise to a low-affinity (deoxy) hemoglobin. HINT shows that the stabilization of the alpha1beta2 interface with mutant Hb Ypsilanti (Asp99alpha-->Tyr) produces a high-affinity (oxy) hemoglobin by reducing hydrophobic-polar contacts in the R state. HINT interaction maps also identified specific sites for mutagenesis at the alpha1beta2 interface that can be explored to shift the allosteric equilibrium in either direction. In addition, the HINT program provides useful diagnostic data for checking the quality of refined crystallographic structures., (Copyright 1997 Academic Press Limited.)

In a case of classical myelomatosis treated with melphalan, a clone of cells with a chromosomal abnormality was found in the bone marrow during remission. There was good reason to think that the hemopoietic cells, rather than plasmacytoma cells, were implicated. Although the clone persisted, no evidence of leukemia developed over a period of observation of 2 yr. The anomaly was interpreted as a duplication of part of the long arm of chromosome 1, which appeared to involve the segment q21 to q31.