Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.

This paper presents the results of experiments that determine the chromosomal location of the mouse gene encoding CCAAT/enhancer binding protein (C/EBP) and measure its expression as a function of tissue type and temporal period of development in mice and rats. Three alleles of the C/EBP gene were identified according to restriction fragment length polymorphisms. The strain distribution pattern of the three alleles was determined in recombinant inbred mouse strains and compared to that of other mouse genes. These results mapped the gene to a position within 2.5 centimorgans (cM) of the structural gene encoding glucose phosphate isomerase on chromosome 7 of the mouse. The expression pattern of the C/EBP gene was studied by a combination of nucleic acid hybridization and antibody staining assays. High levels of C/EBP mRNA were observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates. These included liver, fat, intestine, lung, adrenal gland, and placenta. More detailed analysis of two of these tissues, liver and fat, showed that C/EBP expression was limited to fully differentiated cells. Moreover, analysis of the temporal pattern of expression of C/EBP mRNA in two tissues, liver and intestine, revealed a coordinated induction just prior to birth. These observations raise the possibility that the synthesis of C/EBP may be responsive to humoral factors and that modulation in C/EBP expression might mediate coordinated changes in gene expression that facilitate adaptive challenges met during development or during the fluctuating physiological states of adult life.

The red blood cell (RBC) membrane protein AE1 provides high affinity binding sites for the membrane skeleton, a structure critical to RBC integrity. AE1 biosynthesis is postulated to be required for terminal erythropoiesis and membrane skeleton assembly. We used targeted mutagenesis to assess AE1 function in vivo. RBCs lacking AE1 spontaneously shed membrane vesicles and tubules, leading to severe spherocytosis and hemolysis, but the levels of the major skeleton components, the synthesis of spectrin in mutant erythroblasts, and skeletal architecture are normal or nearly normal. The results indicate that AE1 does not regulate RBC membrane skeleton assembly in vivo but is essential for membrane stability. We postulate that stabilization is achieved through AE1-lipid interactions and that loss of these interactions is a key pathogenic event in hereditary spherocytosis.

We have characterized a new mutant mouse that

Adducins are a family of cytoskeleton proteins encoded by three genes (␣, ␤, ␥). In a comprehensive assay of gene expression, we show the ubiquitous expression of ␣-and ␥-adducins in contrast to the restricted expression of ␤-adducin. ␤-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created ␤-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of ␤-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. ␤-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of ␤-adducin in RBCs leads to decreased membrane incorporation of ␣-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in ␥-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.Adducin was originally described as a protein kinase C substrate in RBCs (1). Purified human RBC adducin consists of two similar polypeptides, ␣ (M r of 103,000) and ␤ (M r of 97,000) (2). In vitro, RBC adducin crosslinks actin filaments with spectrin in a Ca 2ϩ -calmodulin-dependent manner (3), and bundles (4) and caps (5) actin filaments. Adducin is also a substrate for kinase (6, 7) and protein kinase A (8). The third member of the adducin family, ␥-adducin, was discovered as a protein kinase C binding protein in kidney (9). ␥-adducin, a doublet of 84,000 and 86,000 M r , interacts with ␣-adducin in kidney cell extracts (9). Alternatively spliced mRNAs have been described from all three adducin genes (10, 11). Most encode truncated isoforms compared with the originally described isoforms, and their functions are not yet known. To determine the function of ␤-adducin in vivo, we created a null mutation in mice. ␤-adducin null RBCs are osmotically fragile and demonstrate properties similar to RBCs from patients with hereditary spherocytosis. This study demonstrates adducin's importance in RBC structure in vivo. MATERIALS AND METHODSTargeted Disruption of the ␤-Adducin Gene (Add2). The targeting vector was constructed in the pPNT (12) plasmid by using a 3.9-kilobase (kb) EcoRI-BamHI fragment as the 5Ј homology segment and a 1.9-kb EcoRI-NotI fragment as the 3Ј homology segment (Fig. 3A). Transfected 129͞Sv-derived J1 embryonic stem cells (12) were cultured and selected in G418 and gancyclovir. Genomic DNA was digested with EcoRV and was analyzed by Southern blotting using a flanking EcoRVEcoRI fragment as the hybridization probe (Fig. 3B). Blastocyst injection and embryo transfer were performed by using standard techniques (13). Male chimeras were mated to C57BL͞6J females to generate heterozygotes. Progeny were genotyped by using PCR on tail biopsies.Red Blood Cell Analysis. Blood counts were determined by using a Technicon H3 analyzer (Bayer Diagnosti...