Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements.

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.

Mixed synapses discovered and mapped throughout mammalian spinal cord.

Previously, synaptic activity in the spinal cord of adult mammals was attributed exclusively to chemical neurotransmission. In this study, evidence was obtained for the existence, relative abundance, and widespread distribution of "mixed" (chemical and electrical) synapses on neurons throughout the spinal cords of adult mammals. Using combined confocal microscopy and "grid-mapped freeze fracture," 36 mixed synapses containing 88 "micro" gap junctions (median = 45 connexons) were found and mapped to 33 interneurons and motor neurons in Rexed laminae III-IX in cervical, thoracic, and lumbosacral spinal cords of adult male and female rats. Gap junctions were adjacent to presumptive active zones, where even small gap junctions would be expected to increase synaptic efficacy. Two morphological types of mixed synapse were discerned. One type contained distinctive active zones consisting of "nested" concentric toroidal deformations of pre- and postsynaptic membranes, which, because of their unusual topology, were designated as "synaptic sombreros." A second type had gap junctions adjacent to active zones consisting of broad, flat, shallow indentations of the plasma membrane. Morphometric analysis indicates that mixed synapses correspond to 3-5% of all synapses on the somata and proximal dendrites, but, because of their subcellular location and morphology, they could represent 30-100% of excitatory synapses. The relative abundance of mixed synapses on several classes of neurons in spinal cords of adult rats suggests that mixed synapses provide important but previously unrecognized pathways for bidirectional communication between neurons in the mammalian central nervous system.

Mammalian phospholipase D: phosphatidylethanolamine as an essential component.

Bovine kidney phospholipase D (PLD) was assayed by measuring the formation of phosphatidylethanol from added radioactive phosphatidylcholine (PtdCho) in the presence of ethanol, guanosine 5'-[gamma-thio]triphosphate, ammonium sulfate, and cytosol factor that contained small GTP-binding regulatory proteins. The PLD enzyme associated with particulate fractions was solubilized by deoxycholate and partially purified by chromatography on a heparin-Sepharose column. This PLD preferentially used PtdCho as substrate. After purification, the enzyme per se showed little or practically no activity but required an additional factor for the enzymatic reaction. This factor was extracted with chloroform/methanol directly from particulate fractions of various tissues, including kidney, liver, and brain, and identified as phosphatidylethanolamine (PtdEtn), although this phospholipid did not serve as a good substrate. Plasmalogen-rich PtdEtn, dioleoyl-PtdEtn, and L-alpha-palmitoyl-beta-linoleoyl-PtdEtn were effective, but dipalmitoyl-PtdEtn was inert. Sphingomyelin was 30% as active as PtdEtn. The results suggest that mammalian PLD reacts nearly selectively with PtdCho in the form of mixed micelles or membranes with other phospholipids, especially PtdEtn.

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.

Ecdysone-inducible gene expression in mammalian cells and transgenic mice.

During metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy.

cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.

By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel.

Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay.

An expression-cloning strategy was used to isolate a cDNA that encodes a protein that confers calcitonin gene-related peptide (CGRP) responsiveness to Xenopus laevis oocytes. A guinea pig organ of Corti (the mammalian hearing organ) cDNA library was screened by using an assay based on the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR is a chloride channel that is activated upon phosphorylation; this channel activity was used as a sensor for CGRP-induced activation of intracellular kinases. A cDNA library from guinea pig organ of Corti was screened by using this oocyte-CFTR assay. A cDNA was identified that contained an open reading frame coding for a small hydrophilic protein that is presumed to be either a CGRP receptor or a component of a CGRP receptor complex. This CGRP receptor component protein confers CGRP-specific activation to the CFTR assay, as no activation was detected upon application of calcitonin, amylin, neuropeptide Y, vasoactive intestinal peptide, or beta-endorphin. In situ hybridization demonstrated that the CGRP receptor component protein is expressed in outer hair cells of the organ of Corti and is colocalized with CGRP-containing efferent nerve terminals.

A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in the brain.

The Shc adaptor protein, hereafter referred to as ShcA, possesses two distinct phosphotyrosine-recognition modules, a C-terminal Src homology 2 (SH2) domain and an N-terminal phosphotyrosine-binding (PTB) domain, and is itself phosphorylated on tyrosine in response to many extracellular signals. Phosphorylation of human ShcA at Tyr-317 within its central (CH1) region induces binding to the Grb2 SH2 domain and is thereby implicated in activation of the Ras pathway. Two shc-related genes (shcB and shcC) have been identified in the mouse. shcB is closely related to human SCK, while shcC has not yet been found in other organisms. The ShcC protein is predicted to have a C-terminal SH2 domain, a CH1 region with a putative Grb2-binding site, and an N-terminal PTB domain. The ShcC and ShcB SH2 domains bind phosphotyrosine-containing peptides and receptors with a specificity related to, but distinct from, that of the ShcA SH2 domain. The ShcC PTB domain specifically associates in vitro with the autophosphorylated receptors for nerve growth factor and epidermal growth factor. These results indicate that ShcC has functional SH2 and PTB; domains. In contrast to shcA, which is widely expressed, shcC RNA and proteins are predominantly expressed in the adult brain. These results suggest that ShcC may mediate signaling from tyrosine kinases in the nervous system, such as receptors for neurotrophins.

Occurrence of poly(alpha2,8-deaminoneuraminic acid) in mammalian tissues: widespread and developmentally regulated but highly selective expression on glycoproteins.

In tissues of higher organisms homopolymers of alpha2,8-linked N-acetylneuraminic acid can be found as a posttranslational modification on selected proteins. We report here the discovery of homopolymers of alpha2,8-linked deaminoneuraminic acid [poly(alpha2,8-KDN)] in various tissues derived from all three germ layers in vertebrates including mammals. The monoclonal antibody kdn8kdn in conjunction with a bacterial KDNase permitted the detection of poly(alpha2,8-KDN) by immunohistochemistry and immunoblotting. Further evidence for the existence of poly(alpha2,8-KDN) was obtained by gas/liquid chromatography. The poly(alpha2,8-KDN) glycan was detectable in all tissues studied with the exception of mucus-producing cells present in various organs, the extracellular matrix, and basement membranes. However, in certain organs such as muscle, kidney, lung, and brain its expression was developmentally regulated. Despite its widespread tissue distribution, the poly(alpha2,8-KDN) glycan was detected on a single 150-kDa glycoprotein except for a single >350-kDa glycoprotein in kidney, which makes it most distinctive among polysialic acids. The ubiquitous yet selective expression may be indicative of a general function of the poly(alpha2,8-KDN)-bearing glycoproteins.

Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

Evolution of GABAergic circuitry in the mammalian medial geniculate body.

Many features in the mammalian sensory thalamus, such as the types of neurons, their connections, or their neurotransmitters, are conserved in evolution. We found a wide range in the proportion of gamma-aminobutyric acidergic (GABAergic) neurons in the medial geniculate body, from <1% (bat and rat) to 25% or more (cat and monkey). In the bat, some medial geniculate body subdivisions have no GABAergic cells. Species-specific variation also occurs in the somesthetic ventrobasal complex. In contrast, the lateral geniculate body of the visual system has about the same proportion of GABAergic cells in many species. In the central auditory pathway, only the medial geniculate body shows this arrangement; the relative number of GABAergic cells in the inferior colliculus and auditory cortex is similar in each species. The range in the proportion of GABAergic neurons suggests that there are comparative differences in the neural circuitry for thalamic inhibition. We conclude that the number of GABAergic neurons in thalamic sensory nuclei may have evolved independently or divergently in phylogeny. Perhaps these adaptations reflect neurobehavioral requirements for more complex, less stereotyped processing, as in speech-like communication.

Baculovirus-mediated gene transfer into mammalian cells.

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.

Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs.

In mammals, olfactory stimuli are detected by sensory neurons at two distinct sites: the olfactory epithelium (OE) of the nasal cavity and the neuroepithelium of the vomeronasal organ (VNO). While the OE can detect volatile chemicals released from numerous sources, the VNO appears to be specialized to detect pheromones that are emitted by other animals and that convey information of behavioral or physiological importance. The mechanisms underlying sensory transduction in the OE have been well studied and a number of components of the transduction cascade have been cloned. Here, we investigated sensory transduction in the VNO by asking whether VNO neurons express molecules that have been implicated in sensory transduction in the OE. Using in situ hybridization and Northern blot analyses, we found that most of the olfactory transduction components examined, including the guanine nucleotide binding protein alpha subunit (G-alpha-olf), adenylyl cyclase type III, and an olfactory cyclic nucleotide-gated (CNG) channel subunit (oCNC1), are not expressed by VNO sensory neurons. In contrast, VNO neurons do express a second olfactory CNG channel subunit (oCNC2). These results indicate that VNO sensory transduction is distinct from that in the OE but raise the possibility that, like OE sensory transduction, sensory transduction in the VNO might involve cyclic nucleotide-gated ion channels.

Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.

An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liver/bone/kidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents.

Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins.

An enzyme that reduces methionine sulfoxide [Met(O)] residues in proteins [peptide Met(O) reductase (MsrA), EC 1.8.4.6; originally identified in Escherichia coli] was purified from bovine liver, and the cDNA encoding this enzyme was cloned and sequenced. The mammalian homologue of E. coli msrA (also called pmsR) cDNA encodes a protein of 255 amino acids with a calculated molecular mass of 25,846 Da. This protein has 61% identity with the E. coli MsrA throughout a region encompassing a 199-amino acid overlap. The protein has been overexpressed in E. coli and purified to homogeneity. The mammalian recombinant MsrA can use as substrate, proteins containing Met(O) as well as other organic compounds that contain an alkyl sulfoxide group such as N-acetylMet(O), Met(O), and dimethyl sulfoxide. Northern analysis of rat tissue extracts showed that rat msrA mRNA is present in a variety of organs with the highest level found in kidney. This is consistent with the observation that kidney extracts also contained the highest level of enzyme activity.