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.

SummaryTo better characterize aging in mice, the Jackson Aging Center carried out a lifespan study of 31 geneticallydiverse inbred mouse strains housed in a specific pathogen-free facility. Clinical assessments were carried out every 6 months, measuring multiple age-related phenotypes including neuromuscular, kidney and heart function, body composition, bone density, hematology, hormonal levels, and immune system parameters. In a concurrent cross-sectional study of the same 31 strains at 6, 12, and 20 months, more invasive measurements were carried out followed by necropsy to assess apoptosis, DNA repair, chromosome fragility, and histopathology. In this report, which is the initial paper of a series, the study design, median lifespans, and circulating insulinlike growth factor 1 (IGF1) levels at 6, 12, and 18 months are described for the first cohort of 32 females and 32 males of each strain. Survival curves varied dramatically among strains with the median lifespans ranging from 251 to 964 days. Plasma IGF1 levels, which also varied considerably at each time point, showed an inverse correlation with a median lifespan at 6 months (R = )0.33, P = 0.01). This correlation became stronger if the shortlived strains with a median lifespan < 600 days were removed from the analysis (R = )0.53, P < 0.01). These results support the hypothesis that the IGF1 pathway plays a key role in regulating longevity in mice and indicates that common genetic mechanisms may exist for regulating IGF1 levels and lifespan.

Assessment of sleep in mice currently requires initial implantation of chronic electrodes for assessment of electroencephalogram (EEG) and electromyogram (EMG) followed by time to recover from surgery. Hence, it is not ideal for high-throughput screening. To address this deficiency, a method of assessment of sleep and wakefulness in mice has been developed based on assessment of activity/inactivity either by digital video analysis or by breaking infrared beams in the mouse cage. It is based on the algorithm that any episode of continuous inactivity of > or =40 s is predicted to be sleep. The method gives excellent agreement in C57BL/6J male mice with simultaneous assessment of sleep by EEG/EMG recording. The average agreement over 8,640 10-s epochs in 24 h is 92% (n = 7 mice) with agreement in individual mice being 88-94%. Average EEG/EMG determined sleep per 2-h interval across the day was 59.4 min. The estimated mean difference (bias) per 2-h interval between inactivity-defined sleep and EEG/EMG-defined sleep was only 1.0 min (95% confidence interval for mean bias -0.06 to +2.6 min). The standard deviation of differences (precision) was 7.5 min per 2-h interval with 95% limits of agreement ranging from -13.7 to +15.7 min. Although bias significantly varied by time of day (P = 0.0007), the magnitude of time-of-day differences was not large (average bias during lights on and lights off was +5.0 and -3.0 min per 2-h interval, respectively). This method has applications in chemical mutagenesis and for studies of molecular changes in brain with sleep/wakefulness.

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.

The mouse has been a powerful force in elucidating the genetic basis of human physiology and pathophysiology. From its beginnings as the model organism for cancer research and transplantation biology to the present, when dissection of the genetic basis of complex disease is at the forefront of genomics research, an enormous and remarkable mouse resource infrastructure has accumulated. This review summarizes those resources and provides practical guidelines for their use, particularly in the analysis of quantitative traits.

Known vertebrate GATA proteins contain two zinc ®ngers and are required in development, whereas invertebrates express a class of essential proteins containing one GATA-type zinc ®nger. We isolated the gene encoding TRPS1, a vertebrate protein with a single GATA-type zinc ®nger. TRPS1 is highly conserved between Xenopus and mammals, and the human gene is implicated in dominantly inherited tricho-rhino-phalangeal (TRP) syndromes. TRPS1 is a nuclear protein that binds GATA sequences but fails to transactivate a GATA-dependent reporter. Instead, TRPS1 potently and speci®cally represses transcriptional activation mediated by other GATA factors. Repression does not occur from competition for DNA binding and depends on a C-terminal region related to repressive domains found in Ikaros proteins. During mouse development, TRPS1 expression is prominent in sites showing pathology in TRP syndromes, which are thought to result from TRPS1 haploinsuf®ciency. We show instead that truncating mutations identi®ed in patients encode dominant inhibitors of wild-type TRPS1 function, suggesting an alternative mechanism for the disease. TRPS1 is the ®rst example of a GATA protein with intrinsic transcriptional repression activity and possibly a negative regulator of GATAdependent processes in vertebrate development.

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...

Studies of mouse models of anemia have long provided fundamental insights into red blood cell formation and function. Here we show that the semidominant mouse mutation Nan (“neonatal anemia”) carries a single amino acid change (E339D) within the second zinc finger of the erythroid Krüppel-like factor (EKLF), a critical erythroid regulatory transcription factor. The mutation alters the DNA-binding specificity of EKLF so that it no longer binds promoters of a subset of its DNA targets. Remarkably, even when mutant Nan and wild-type EKLF alleles are expressed at equivalent levels, the mutant form selectively interferes with expression of EKLF target genes whose promoter elements it no longer binds. This interference yields a distorted genetic output and selective protein deficiencies that differ from those seen in EKLF-heterozygous and EKLF-null red blood cells and presents a unique and unexpected mechanism of inherited disease.