Cloning and characterization of soluble and transmembrane isoforms of a novel component of the murine type I interferon receptor, IFNAR 2.

This report describes the cloning of cDNAs encoding transmembrane and soluble isoforms of a novel chain of the murine type I interferon (IFN) receptor and characterization of its capability to bind ligand and transduce signals. The transmembrane receptor (murine IFNAR 2c) has an extracellular domain of 215 amino acids and an intracellular domain of 250 amino acids, with 48% amino acid and 71% nucleotide identity with human IFNAR 2c. The cDNA for the soluble murine receptor (IFNAR 2a) encodes a 221-amino acid polypeptide identical to the first 210 amino acids of IFNAR 2c plus a novel 11 amino acids. Northern blot analyses show that murine IFNAR 2 is expressed as two transcripts of 4 kilobases encoding the transmembrane isoform and 1.5 kilobases encoding the more abundant soluble isoform. Studies using primary murine cells that lack IFNAR 1 show that IFNAR 2 is expressed, and cells bind type I IFN ligand, but do not transduce signals as detected by electrophoretic mobility shift assays of ISGF3 or GAF complexes binding to their cognate oligonucleotides. These cells show no effects on the ability of IFNgamma to activate these complexes. These studies demonstrate that the IFNAR 2 transmembrane (2c) and soluble (2a) isoforms are conserved between the human and mouse and that IFNAR 2c has intrinsic ligand binding activity, but no intrinsic signal transducing activity as measured in this study.

A type I interferon signaling factor, ISF21, encoded on chromosome 21 is distinct from receptor components and their down-regulation and Is necessary for transcriptional activation of interferon-regulated genes.

The type I interferons (IFNs) are a family of cytokines, comprising at least 17 subtypes, which exert pleiotropic actions by interaction with a multi-component cell surface receptor and at least one well characterized signal transduction pathway involving JAK/STAT (Janus kinase/signal transducer and activator of transcription) proteins. In a previous report, we showed that a signaling factor, encoded by a gene located on the distal portion of chromosome 21, distinct from the IFNAR-1 receptor, was necessary for 2'-5'-oligoadenylate synthetase activity and antiviral responses, but not for high affinity ligand binding. In the present studies using hybrid Chinese hamster ovary cell lines containing portions of human chromosome 21, we show that the type I IFN signaling molecule, designated herein as ISF21, is distinct from the second receptor component, IFNAR-2, which is expressed in signaling and non-signaling cell lines. The location of the gene encoding ISF21 is narrowed to a region between the 10;21 and the r21 breakpoints, importantly eliminating the Mx gene located at 21q22.3 (the product of which is involved in IFN-induced antiviral responses) as a candidate for the signaling factor. To characterize the action of this factor in the type I IFN signaling pathway, we show that it acts independently of receptor down-regulation following ligand binding, both of which occur equally in the presence or absence of the factor. In addition, we demonstrate that ISF21 is necessary for transcriptional activation of 2'-5'-oligoadenylate synthetase, 6-16, and guanylate-binding protein gene promoter reporter constructs, which are mediated by several signaling pathways. ISF21 represents a novel factor as the localization to chromosome 21, and the data presented in this study exclude any of the known type I IFN signal-transducing molecules.

A high observed substitution rate in the human mitochondrial DNA control region.

The rate and pattern of sequence substitutions in the mitochondrial DNA (mtDNA) control region (CR) is of central importance to studies of human evolution and to forensic identity testing. Here, we report a direct measurement of the intergenerational substitution rate in the human CR. We compared DNA sequences of two CR hypervariable segments from close maternal relatives, from 134 independent mtDNA lineages spanning 327 generational events. Ten substitutions were observed, resulting in an empirical rate of 1/33 generations, or 2.5/site/Myr. This is roughly twenty-fold higher than estimates derived from phylogenetic analyses. This disparity cannot be accounted for simply by substitutions at mutational hot spots, suggesting additional factors that produce the discrepancy between very near-term and long-term apparent rates of sequence divergence. The data also indicate that extremely rapid segregation of CR sequence variants between generations is common in humans, with a very small mtDNA bottleneck. These results have implications for forensic applications and studies of human evolution.

Binding of interferon-alpha and -beta to a component of the human type I interferon receptor expressed in simian cells.

The type I interferons (IFNs) are a family of homologous cytokines that compete for receptor binding. Past experiments with a cloned human IFN-alpha receptor component (designated herein as HuIFNAR-1) transfected into different cell backgrounds have given contradictory results in terms of binding and signalling after exposure of cells to different human type I IFNs. In order to investigate the binding specificity of human type I IFN subtypes to HuIFNAR-1, a cDNA encoding HuIFNAR-1 was transfected into simian COS cells. HuIFNAR-1 expression in COS cells, which was confirmed by Northern blot analysis, resulted in increased binding of 125I-labelled HuIFN-alpha 2 and -beta. These data support the participation of this receptor component in ligand binding, probably in association with other receptor components.

Isolation and characterization of the versicolorin B synthase gene from Aspergillus parasiticus. Expansion of the aflatoxin b1 biosynthetic gene cluster.

Versicolorin B synthase catalyzes the side chain cyclization of racemic versiconal hemiacetal to the bisfuran ring system of(-)-versicolorin B, an essential transformation in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. The dihydrobisfuran is key to the mutagenic nature of aflatoxin B1. The protein, which shows 58% similarity and 38% identity with glucose oxidase from Aspergillus niger, possesses an amino-terminal sequence homologous to the ADP-binding region of other flavoenzymes. However, this enzyme does not require flavin or nicotinamide cofactors for its cyclase activity. The 643-amino acid native enzyme contains three potential sites for N-linked glycosylation, Asn-Xaa-Thr or Asn-Xaa-Ser. The cDNA and genomic clones of versicolorin B synthase were isolated by screening the respective libraries with random-primed DNA probes generated from an exact copy of an internal vbs sequence. This probe was created through polymerase chain reaction by using nondegenerate polymerase chain reaction primers derived from the amino acid sequences of peptide fragments of the enzyme. The 1985-base genomic vbs DNA sequence is interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing, and detailed restriction mapping of the vbs-containing genomic clones revealed the presence of omtA, a methyltransferase active in the biosynthesis, 3.3 kilobases upstream of vbs and oriented in the opposite direction from vbs. The presence of omtA in close proximity to vbs supports the theory that the genes encoding the aflatoxin biosynthetic enzymes in A. parasiticus are clustered.

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.

Estimating energy needs of pediatric patients with burns.

Recent reports indicate that formulas used to estimate the energy needs of pediatric patients with burns overestimate the energy consumption that results in maintenance of body weight in these patients. The purpose of this project was to determine which of four formulas (identified as Boston, Galveston, UCSD original, or UCSD modified) most accurately estimated the energy consumption needed to maintain body weight in pediatric patients with burns. The medical records of 12 subjects, 1 to 12 years of age, who maintained body weight (within +/- 10% of dry weight) were reviewed retrospectively. Analysis of variance was used to compare the patients' energy intakes with their estimated energy needs by use of each of the four formulas. All four formulas overestimated the energy intakes that resulted in maintenance of body weight (p < 0.05), but the estimate that used the Galveston formula was closest to actual energy consumption.

A tightly bound chromosome antigen is detected by monoclonal antibodies in a ring-like structure on human centromeres.

Monoclonal antibodies (Mabs) were raised against isolated Chinese hamster protein-depleted chromosomes Chromosome scaffolds) in order to probe for components involved in the higher-order structure of mammalian chromosomes. One of the Mabs detected a ring-like structure in metaphase at the centromere, which is conserved between Chinese hamster and human cells. Additionally, the Mab stained the centrioles in interphase cells in these two species. The antigen was enriched in chromosomal protein preparations by comparison with nuclear protein samples, and has an apparent Mr = 170,000. The centromere antigen remained present in chromosome scaffold preparations, indicating that it was tightly associated with DNA. The antigen was distinct in its centromeric localisation from any of the centromere antigens reported to date. A possible role of the antigen in stabilising the centromere, by holding the sister chromatids together until their separation at the metaphase-anaphase transition is presented.

A gene on human chromosome 21 located in the region 21q22.2 to 21q22.3 encodes a factor necessary for signal transduction and antiviral response to type I interferons.

The type I interferons (IFNs) are a family of multifunctional cytokines which includes the 15 IFN alpha subtypes and IFN beta. These IFNs compete for binding to cell surface receptors. However, murine cells transfected with a cDNA for a human IFN alpha receptor (IFNAR) developed an antiviral response only to human IFN alpha B, but not to human IFN alpha 2 nor -beta(1). In this study we show, using a panel of CHO-human chromosome 21 hybrid cell lines which all express IFNAR, that only those containing the region 21q22.2 to 21q22.3 transduce signals for IFN responses. Two such hybrid cell lines responded to IFNs alpha 2, -alpha B and -beta by induction of 2'-5' oligoadenylate synthetase and resistance to viral infection. Other hybrid cell lines, that lacked the region 21q22.2-3, failed to transduce signals as above; even though they expressed IFNAR and bound human IFN alpha 2, -alpha B, and -beta. These data demonstrate that a gene(s) located in the region 21q22.2-3 encodes a factor(s) which is necessary for signaling but does not influence ligand binding. This factor is not the cofactor required for IFN gamma signaling which is located in the region 21p to 21q22.1(2).