Saccharomyces cerevisiae takes up siderophorebound iron through two distinct systems, one that requires siderophore transporters of the ARN family and one that requires the high affinity ferrous iron transporter on the plasma membrane. Uptake through the plasma membrane ferrous iron transporter requires that the iron first must dissociate from the siderophore and undergo reduction to the ferrous form. FRE1 and FRE2 encode cell surface metalloreductases that are required for reduction and uptake of free ferric iron. The yeast genome contains five additional FRE1 and FRE2 homologues, four of which are regulated by iron and the major iron-dependent transcription factor, Aft1p, but whose function remains unknown. Fre3p was required for the reduction and uptake of ferrioxamine B-iron and for growth on ferrioxamine B, ferrichrome, triacetylfusarinine C, and rhodotorulic acid in the absence of Fre1p and Fre2p. By indirect immunofluorescence, Fre3p was expressed on the plasma membrane in a pattern similar to that of Fet3p, a component of the high affinity ferrous transporter. Enterobactin, a catecholate siderophore, was not a substrate for Fre3p, and reductive uptake required either Fre1p or Fre2p. Fre4p could facilitate utilization of rhodotorulic acid-iron when the siderophore was present in higher concentrations. We propose that Fre3p and Fre4p are siderophore-iron reductases and that the apparent redundancy of the FRE genes confers the capacity to utilize iron from a variety of siderophore sources.Virtually every organism on earth requires iron as an essential nutrient. Although iron is the second most abundant metal in the crust of the earth, the bioavailability of iron can be extremely low. This poor bioavailability occurs because iron is rapidly oxidized in an aerobic environment to the ferric form (Fe(III)), 1 which is poorly soluble in water and forms precipitates of oxyhydroxides. Microorganisms have the capacity to scavenge iron from insoluble precipitates by secreting and taking up siderophores, low molecular weight compounds that bind to Fe(III) with very high affinity and specificity. Siderophores are synthesized and secreted in the iron-free form, which then binds and solubilizes Fe(III) in the extracellular environment. The Fe(III)-siderophore complex is then recognized and selectively taken up by specific transport mechanisms. Many microorganisms synthesize one or a few types of siderophores, yet have the capacity to take up iron from a variety of siderophores secreted by other species of bacteria and fungi (1). Budding and fission yeast appear to be an exception; they neither synthesize nor secrete these compounds (2, 3). Saccharomyces cerevisiae can, however, recognize and take up iron from a variety of structurally distinct siderophores (4 -10).S. cerevisiae has two genetically separable systems for the uptake of siderophore-bound iron. One system depends on a family of homologous transporters of the major facilitator superfamily that is expressed as part of the AFT1 regulon and are termed ARN1, ARN2...

We examined the release of cardiac troponin T (cTnT) and I (cTnI) into the blood of patients after acute myocardial infarction (AMI). Three postAMI serum samples were applied in separate analytical runs onto a calibrated gel filtration column (Sephacryl S-200), and the proteins were separated by molecular weight. Using commercial cTnT and cTnI assays measured on collected fractions, we found that troponin was released into blood as a ternary complex of cTnT-I-C, a binary complex of cTnI-C, and free cTnT, with no free cTnI within the limits of the analytical methodologies. The serum samples were also examined after incubation with EDTA and heparin. EDTA broke up troponin complexes into individual subunits, whereas heparin had no effect on the assays tested. We added free cTnC subunits to 24 AMI serum samples and found no marked increase in the total cTnI concentrations, using an immunoassay that gave higher values for the cTnI-C complex than free cTnI. To characterize the cross-reactivity of cTnT and cTnI assays, purified troponin standards in nine different forms were prepared, added to serum and plasma pools, and tested in nine quantitative commercial and pre-market assays for cTnI and one approved assay for cTnT. All nine cTnI assays recognized each of the troponin I forms (complexed and free). In five of these assays, the relative responses for cTnI were nearly equimolar. For the remainder, the response was substantially greater for complexed cTnI than for free cTnI. Moreover, there was a substantial difference in the absolute concentration of results between cTnI assays. The commercial cTnT assay recognized binary and ternary complexes of troponin on a near equimolar basis. We conclude that all assays are useful for detection of cardiac injury. However, there are differences in absolute cTnI results due to a lack of mass standardization and heterogeneity in the cross-reactivities of antibodies to various troponin I forms.

A major proportion of carcinomas induced by mouse mammary tumour virus (MMTV) show evidence for proviral activation of a cellular gene, int-2, on chromosome 7. The sequence of 7869 bp of DNA spanning the transcription unit of int-2 was determined and compared with that of a series of int-2-specific cDNA clones derived from mammary tumour RNA. The predicted positions of intron-exon boundaries, established by alignment of cDNA and chromosomal DNA sequences, indicate that the gene comprises at least three exons. An open reading frame capable of encoding a protein of 245 amino acids with an estimated mol. wt of 27 kd, is flanked by substantial non-coding segments at both 5' and 3' ends. Comparison of the chromosomal DNA sequence and the predicted amino acid sequence with available data-bases has revealed no homology to other known genes. These results are discussed in relation to the status of int-2 as a candidate proto-oncogene.

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We sequenced two recombinant DNA clones constituting a single provirus of the milk-transmitted mouse mammary tumor virus characteristic of BR6 mice. The complete provirus is 9,901 base pairs long, flanked by 6 base-pair duplications of cellular DNA at the site of integration. Five extensive blocks of open reading frame corresponding to the gag gene, the presumed protease, the pol and env genes, and the open reading frame orf within the long terminal repeat of the provirus were readily discernible. Translation of gag, protease, and pol involved three different translational reading frames to produce the three overlapping polyprotein precursors Pr77, Pr110, and Prl60 found in virus-infected cells. Synthesis of the reverse transcriptase and endonuclease therefore required two separate frameshifts to suppress the termination codons at the ends of the Pr77 and PrllO domains. Direct evidence is presented for translational readthrough of both stop codons in an in vitro protein synthesis system.