Betty Josephson

The position of integration affects expression of the Aγ-globin-encoding gene linked to HS3 in transgenic mice

Proper expression of the human beta-globin (beta Glb) locus is dependent on the presence of a major regulatory element located upstream from the beta Glb gene cluster, the locus control region (LCR). The LCR, as well as the individual DNase-I-hypersensitive sites from which it is composed, have been shown to provide position-of-integration-independent expression in transgenic mice. Here, we report that a transgenic founder carrying multiple integrations of a hypersensitive site 3::A gamma globin gene (HS3::A gamma) construct produced three types of progeny, one with zero A gamma expression in the adult stage, one with minimal A gamma expression (1% of A gamma-expressing cells) and one with abundant A gamma expression (100% A gamma-expressing cells). The possibility that these phenotypes were due to parental imprinting or to DNA rearrangements of the transgene or to point mutations of the HS3 core or the A gamma promoter were excluded. The pattern of inheritance of the three HS3::A gamma transgene phenotypes indicate that the transgene has integrated into three different chromosomes. These results provide direct evidence that the HS3 of the LCR is not sufficient to protect the A gamma gene from position effects excerted by the surrounding chromatin.

A highly efficient chemical isolation procedure for the rat placental transferrin receptor.

A chemical method for the purification of rat placental transferrin receptor is described. After initial solubilization and concentration by ammonium sulfate precipitation, radioiron-tagged diferric transferrin was added to the dialyzed receptor fraction and subjected to anion-exchange chromatography on DEAE-Sephacel. Elution with a Tris-HCl buffer gradient yields a single fraction of radioactivity containing both free transferrin and the receptor-transferrin as a complex. Further separation of the receptor-transferrin complex from the free transferrin is achieved by gel chromatography on a AcA34-Sepharose 6B separation system. Final purification is obtained by preparative gel electrophoresis in 5% polyacrylamide gels. The receptor was shown to be pure by various methods including HPLC chromatography. The average yield was 20-30 mg receptor-transferrin complex/100 g placental tissue. Because of the purely chemical approach, this method is universally applicable for the isolation of transferrin receptors from various tissues.

Basis of Plasma Iron Exchange in the Rabbit

Rabbit transferrin in vitro is shown to load ferrous iron at random on its specific binding sites. The release of iron to reticulocytes is shown to be an all-or-none phenomenon. The two monoferric transferrins have similar in vivo plasma iron clearance rates and tissue distribution. Diferric transferrin, while giving a similar tissue distribution of radioiron, has a plasma iron clearance rate approximately twice that of the monoferric transferrins at low plasma iron concentrations. This difference diminishes as the plasma iron concentration increases. These results are consistent with a progressively greater in vivo conversion of mono- to diferric transferrin as transferrin saturation increases. The in vivo plasma iron turnover in the rabbit increases progressively as the plasma iron increases, from a mean value of approximately 0.8 mg/dl whole blood per d at a plasma iron concentration of 50 mug/dl to 2.0 at a plasma iron concentration of 300. The molecular behavior of transferrin and its iron over this range was investigated using (125)I-transferrin, [(55)Fe]monoferric transferrin, and [(59)Fe]diferric transferrin. The equilibrium distribution of transferrin between its apo-, mono-, and diferric moieties was similar to that predicted on the basis of the percent saturation and random distribution. Rate constants of iron loading and unloading calculated from the percent saturation and from the clearance rates of [(55)Fe]monoferric and [(59)Fe]diferric transferrin were similar to those derived from changes in injected (125)I-apotransferrin. On the basis of these data, it is concluded that the plasma transferrin pool is nonhomogeneous and that the relative size of the mono- and diferric cycles depends on transferrin saturation. A formula is proposed for correcting the plasma iron turnover, thereby eliminating the effect of plasma iron concentration, so as to reflect directly the number of tissue transferrin receptors.