p47phox Phox Homology Domain Regulates Plasma Membrane but Not Phagosome Neutrophil NADPH Oxidase Activation

The assembly of cytosolic subunits p47^phox, p67^phox, and p40^phox with flavocytochrome b₅₅₈ at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47^phox subunit plays a critical role in oxidase assembly. Recent studies showed that the p47^phox Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47^phox PX domain in neutrophils is unclear, we investigated its role using p47^phox knock-out (KO) mouse neutrophils to express human p47^phox and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47^phox proteins were expressed at levels similar to endogenous murine p47^phox, with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47^phox rescued p47^phox KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47^phox with Arg⁹⁰ substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47^phox Arg⁹⁰ mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47^phox PX domain for neutrophil NADPH oxidase activation.     

Structure of the rat p53 tumor suppressor gene.

Aberration within the p53 tumor suppressor gene is the most frequently identified genetic damage in human cancer. Regulatory functions proposed for the p53 protein include modulation of the cell cycle, cellular differentiation, signal transduction, and gene expression. Additionally, the p53 gene product may guard the genome against incorporation of damaged DNA. To facilitate study of its role in carcinogenesis using a common animal model, we determined the structure of the rat p53 gene. We identified 18 splice sites and defined 25 bases of the intervening sequences adjacent to these sites. We also discovered an allelic polymorphism that occurs within intron 5 of the gene. The rat gene approximates the mouse ortholog. It is 12 kb in length with the non-coding exon 1 separated from exon 2 by 6.2 kb of intervening sequence. The location and size of all rat gene introns approximate those of the mouse. Whereas the mouse and human genes each contain 11 exons, the rat p53 gene is composed of only 10. No intervening sequence occurs between the region of the rat gene corresponding to exons 6 and 7 of the mouse and human p53 genes. This implies intron 6 may be functionally insignificant for species in which it is retained. To extrapolate to p53 involvement in human tumorigenesis, we suggest that mutational events within intron 6 may not be of pathological significance unless splicing is hindered.     

Orthologues of the Caenorhabditis elegans Longevity Gene clk-1 in Mouse and Human

The clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a biological role in longevity (Ewbank et al., 1997, Science 275: 980-983). The primary structure of CLK-1 showed a significant homology to Saccharomyces cerevisiae Coq7p/Cat5p, which is required for the biosynthesis of ubiquinone and the derepression of gluconeogenic genes. In the present study, we isolated and characterized human and mouse orthologues of the COQ7/CLK-1 gene. Sequence analysis of both the human and the mouse COQ7 cDNAs showed an open reading frame composed of 217 amino acids with calculated molecular mass of 24,309 and 24,044 Da, respectively. Homology search revealed that human COQ7 showed 85% identity to mouse COQ7, 89% identity to rat COQ7, 53% identity to C. elegans CLK-1, and 37% identity to S. cerevisiae Coq7p/Cat5p. Zoo blot analysis implied that the COQ7 gene was well conserved among mammal, bird, and reptile genomes. Tissue blot analysis showed that human COQ7 is dominantly transcribed in heart and skeletal muscle. Genomic analyses revealed that the human COQ7 gene is composed of six exons spanning 11 kb of human genome as a single-copy gene. Radiation hybrid mapping assigned the COQ7 gene to human chromosome 16p12.3-p13.11.     

Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47phox-deficient chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O₂^?) and other microbicidal oxidants due to mutations in one of the five components of the O₂^?-generating NADPH oxidase complex. The most common autosomal subtype of CGD is caused by mutations in NCF1, encoding the NADPH subunit p47^phox. Usually, these mutations are the result of unequal exchange of chromatid between NCF1 and one of its two pseudogenes. We have now investigated in detail the breakpoints within or between these (pseudo) NCF1 genes in 43 families with p47^phox-deficient CGD by means of multiplex ligase-dependent probe amplification (MLPA). In 24 families the patients totally lacked NCF1 sequences, indicating that in these families the cross-over points are located between NCF1 and its pseudogenes. Six other families were compound heterozygous for a total NCF1 deletion and another mutation in NCF1 on the other allele. In 8 families, the patients lacked NCF1 exons 1–4 but had retained NCF1 exons 6–10, indicating that a cross-over point is located within NCF1 between exons 4 and 6. Similarly, in 4 families a cross-over point was located within NCF1 between exons 2 and 4. Similar cross-overs, in heterozygous form, were observed in family members of the patients. Several patients were compound heterozygous for total and partial NCF1 deletions. Thus, at least three different cross-over points exist within the NCF1 gene cluster, indicating that autosomal p47^phox-deficient CGD is genetically heterogeneous but can be dissected in detail by MLPA.     

The Structure and Expression of the Murine Wildtype p53-Induced Phosphatase 1 (Wip1) Gene

The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5′-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11.     

Characterization of the gene encoding murine heparin-binding epidermal growth factor-like growth factor

The mouse gene (mHB-EGF) encoding heparin-binding epidermal growth factor-like growth factor was isolated from a mouse 129SVJ genomic library. DNA sequence analysis confirmed that the clone contained six exons (I–VI) and five introns (A–E), and spanned approx. 14 kb of DNA. PCR analysis showed that introns A–E of mHB-EGF are 203 bp, 2.5 kb, 5.5 kb, 825 bp and 272 bp in length, respectively. These results establish that mHB-EGF is similar in organization to human HB-EGF (hHB-EGF). However, DNA sequence analysis of introns A–E of mHB-EGF failed to show significant overall homology with those of hHB-EGF     

Drug-selected complete restoration of superoxide generation in Epstein-Barr virus-transformed B cells from p47 phox -deficient chronic granulomatous disease patients by using a bicistronic retrovirus vector encoding a human multi-drug resistance gene (MDR1) and the p47 phox gene

Chronic granulomatous disease (CGD) is a group of disorders characterized by the failure of phagocytes to produce superoxide. One-third of the cases of CGD in the USA and Europe results from defects in the gene encoding p47 ^phox , a cytoplasmic component of NADPH oxidase for superoxide generation. In this study, we constructed the bicistronic retrovirus vector Ha-MDR-IRES-p47, which carries cDNAs for a human multi-drug-resistance gene (MDR1) and p47 ^phox . The amphotropic retroviral producer cells were generated, and the supernatant of the producer cells was used to transduce Epstein-Barr virus-transformed B (EBV-B) cells, established from B cells of p47 ^phox -deficient CGD patients, as an in vitro model of gene therapy for p47 ^phox -deficient CGD. The transduced cells expressed both P-glycoprotein and p47 ^phox protein, and the expression levels were increased after appropriate vincristine selection. The levels of superoxide production in the vincristine-selected cells were increased to a level similar to normal EBV-B cells. This result suggests that it is possible to achieve 100% correction of the CGD defect in p47 ^phox -deficient EBV-B cells by using the bicistronic vector. This strategy could be employed not only in vitro, but also in vivo, in the gene therapy of a number of inherited diseases. Received: 8 June 1998 / Accepted: 5 August 1998     

Mouse and Human Neuronal Pentraxin 1 (NPTX1): Conservation, Genomic Structure, and Chromosomal Localization

We have previously identified novel members of the pentraxin family (neuronal pentraxin 1 and 2) that are expressed in the nervous system. Neuronal pentraxin 1 (NP1) was identified as a rat protein that may mediate the uptake of synaptic material and the presynaptic snake venom toxin, taipoxin. NP2 was identified as a separate gene discovered by screening for a human homolog for NP1. Here, we report human cDNA and mouse genomic DNA sequences for NP1 (gene symbol NPTX1). Human NP1 and mouse NP1 show 95 and 99% amino acid identity, respectively, with rat NP1 and conserve all potential glycosylation sites. Like rat NP1, human NP1 message is large (6.5 kb) and is exclusively localized to the nervous system. The mouse NP1 gene is 13 kb in length and contains four introns that break the coding sequence of NP1 in the same positions as the introns of the human NP2 gene. The human and mouse NP1 genes are localized to chromosome 17q25.1–q25.2 and chromosome 11e2–e1.3, respectively. These data demonstrate the existence of a separate family of pentraxin proteins that are expressed in the human brain and other tissues and that may play important roles in the uptake of extracellular material.     

Localization of a Human Homolog of the Mouse Pericentrin Gene (PCNT) to Chromosome 21qter

Exon trapping was used to identify portions of genes from cosmid DNA of a human chromosome 21-specific library LL21NC02-Q. More than 650 potential exons have been cloned and characterized to date. Among these, 3 trapped “exons” showed strong homology to different regions of the cDNA for the mouse pericentrin (Pcnt) gene (Doxseyet al., Cell76: 639–650, 1994), indicating that these 3 exons are portions of a human homolog of the mouse pericentrin gene. With PCR amplification, Southern blot analysis, and FISH, we have mapped this presumed human pericentrin gene (PCNT) to the long arm of chromosome 21 between marker PFKL and 21qter. Pericentrin is a conserved protein component of the filamentous matrix of the centrosome involved in the initial establishment of the organized microtubule array. No candidate hereditary disorder for pericentrin deficiency/abnormality has yet been mapped in the most distal region of 21q; in addition the role of triplication of the pericentrin gene in the pathophysiology or etiology of trisomy 21 is currently unknown.