Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation.

The Atp7b protein is a copper-transporting ATPase expressed predominantly in the liver and to a lesser extent in most other tissues. Mutations in the ATP7B gene lead to Wilson disease, a copper toxicity disorder characterized by dramatic build-up of intracellular hepatic copper with subsequent hepatic and neuro-logical abnormalities. Using homologous recombination to disrupt the normal translation of ATP7B, we have generated a strain of mice that are homozygous mutants (null) for the Wilson disease gene. The ATP7B null mice display a gradual accumulation of hepatic copper that increases to a level 60-fold greater than normal by 5 months of age. An increase in copper concentration was also observed in the kidney, brain, placenta and lactating mammary glands of homo-zygous mutants, although milk from the mutant glands was copper deficient. Morphological abnormalities resembling cirrhosis developed in the majority of the livers from homozygous mutants older than 7 months of age. Progeny of the homozygous mutant females demonstrated neurological abnormalities and growth retardation characteristic of copper deficiency. Copper concentration in the livers of the newborn homozygous null mutants was decreased dramatically. In summary, inactivation of the murine ATP7B gene produces a form of cirrhotic liver disease that resembles Wilson disease in humans and the 'toxic milk' phenotype in the mouse.

Molecular cloning of ictacalcin: a novel calcium-binding protein from the channel catfish, Ictalurus punctatus.

Calcium is essential for a variety of functions in animals, including signal transduction, transmission of nerve impulses, and bone and scale growth. In freshwater adapted teleosts, blood calcium levels are maintained constant (2-4 mM) even at low external calcium concentration (< 0.01 mM). Epithelial cells in skin and gill have been implicated in calcium homeostasis. We have cloned a cDNA from Ictalurus punctatus, the channel catfish, that codes for ictacalcin, a novel member of the S100 family of calcium-binding protein. In-situ hybridization demonstrates ictacalcin mRNA is abundant in epithelial cells of olfactory rosette, barbel, skin and gill but not brain or muscle. The presence of ictacalcin protein in these tissues was confirmed by immuno-blot analysis. Tissue extracts and recombinant ictacalcin bind calcium with attendant changes in electrophoretic mobility indicative of changes in protein conformation. The calcium-binding activity and abundant localization of ictacalcin in epithelial cells of several tissues indicates that this protein plays an important role in catfish calcium homeostasis.

Olfactory marker protein (OMP) gene deletion causes altered physiological activity of olfactory sensory neurons.

Olfactory marker protein (OMP) is an abundant, phylogentically conserved, cytoplasmic protein of unknown function expressed almost exclusively in mature olfactory sensory neurons. To address its function, we generated OMP-deficient mice by gene targeting in embryonic stem cells. We report that these OMP-null mice are compromised in their ability to respond to odor stimull, providing insight to OMP function. The maximal electroolfactogram response of the olfactory neuroepithelium to several odorants was 20-40% smaller in the mutants compared with controls. In addition, the onset and recovery kinetics following isoamyl acetate stimulation are prolonged in the null mice. Furthermore, the ability of the mutants to respond to the second odor pulse of a pair is impaired, over a range of concentrations, compared with controls. These results imply that neural activity directed toward the olfactory bulb is also reduced. The bulbar phenotype observed in the OMP-null mouse is consistent with this hypothesis. Bulbar activity of tyrosine hydroxylase, the rate limiting enzyme of catecholamine biosynthesis, and content of the neuropeptide cholecystokinin are reduced by 65% and 50%, respectively. This similarity to postsynaptic changes in gene expression induced by peripheral olfactory deafferentation or naris blockade confirms that functional neural activity is reduced in both the olfactory neuroepithelium and the olfactory nerve projection to the bulb in the OMP-null mouse. These observations provide strong support for the conclusion that OMP is a novel modulatory component of the odor detection/signal transduction cascade.

Expression of the human Achaete-scute 1 gene in olfactory neuroblastoma (esthesioneuroblastoma).

Olfactory neuroblastoma (ONB) is a rare neuronal malignancy of the olfactory mucosal. Markers used in the diagnosis of ONB do not distinguish ONB from other neuronal tumors or tumors with neuroendocrine features thus making the diagnosis of ONB difficult. Using a modified RT-PCR technique, we show that the human homologue of the Drosophila achaete-scute gene HASH1 is expressed in 6 primary and one metastatic ONB specimens, whereas Olfactory Marker Protein (OMP) is not. Previous studies have shown that HASH1 is expressed in immature olfactory neurons and is required for their development. OMP, whose function is unknown, is expressed exclusively in mature olfactory neurons. Together, these data suggest that ONB is derived from immature olfactory neurons of neuroectodermal origin. Analysis of RNA expression in primary tumor specimens and in an established cell line make this an ideal system to study olfactory growth and differentiation. Furthermore, these studies represent the first molecular genetic analysis of this rare and unusual neuronal tumor.

Human olfactory receptor neurons contain OMP mRNA in their dendritic and axonal processes.

The cellular expression of olfactory marker protein (OMP) mRNA and protein was investigated in the olfactory mucosa of humans ranging in age from 26 weeks of gestation to 85 years using in situ hybridization and immunocytochemistry. OMP mRNA and protein were most abundant in the somas of olfactory receptor neurons (ORNs). The hybridization signal over the ORN somal layer was greater in older subjects than in younger ones, reflecting either a higher neuronal density or more OMP mRNA per cell. In contrast, it was significantly lower in subjects with Alzheimer's disease when compared with an age-matched control. Characteristics of older subjects were patchiness in the distribution of OMP-expressing ORNs and the occurrence of subepithelial invaginations containing OMP-positive neurons. In addition, a significant hybridization signal was detected in the apical olfactory epithelium containing the dendrites, dendritic knobs, and cilia of ORNS, and over olfactory nerve bundles in the lamina propria, indicating the occurrence of OMP mRNA in dendritic and axonal domains.

Human and rodent OMP genes: conservation of structural and regulatory motifs and cellular localization.

Immunocytochemical analysis has demonstrated that expression of the olfactory marker protein (OMP) is highly restricted to mature olfactory receptor neurons in virtually all vertebrate species from fish to man. We have now cloned the OMP gene from human and mouse and demonstrated conservation of gene structure, protein sequence, and Olf-1 and upstream binding region (UBE) regulatory domains. The OMP gene in all species studied lacks canonical TATA and CAAT motifs and introns. The deduced protein sequence is 88.4% identical between mouse and human, and most of the differences observed are conservative changes. The proximal Olf-1 binding sites differ by two purine-purine replacements and effectively cross-compete in mobility shift assays. The distal Olf-1 binding site is also highly conserved in terms of both sequence and binding activity. The availability of sequence from multiple species has permitted us to determine that the UBE site has close similarity to motifs that bind members of the NF-1 family of transcription factors. Gel mobility shift assays confirm this prediction, providing additional insight into mechanisms that may participate in the stringent regulation of the expression of this neuronal-specific protein. Furthermore, we demonstrate the in situ localization of OMP mRNA in human olfactory neuro-epithelium and its colocalization to immunocytochemically identified human olfactory receptor neurons.

[Isolation and analysis of brain-specific sequences from cDNA libraries for various segments of the human brain]. / Vydelenie i analiz mozgospetsificheskikh posledovatel'nostei iz bibliotek kDNK raznykh otedelov mozga cheloveka.

The cDNA libraries in gt10 were constructed from total poly(A)+RNA of human forebrain cortex, cerebellar cortex and medulla oblongata. We selected the clones which gave hybridization signal with brain cDNA only, or gave no signal from these libraries. Expression pattern and structure of two brain-specific clones Hfb1 from forebrain library and Hmob3 from medulla oblongata library were analyzed in detail. Hfb1 hybridized to two different transcripts (about 5 and 2 kb) from frontal cortex, but to a single (longest) from cerebellum. Hfb1 sequence includes 958 nucleotides. Comparison of Hfb1 with the Gene Bank revealed no homology with the sequences present in the Bank. At 3'-end there is poly(A) tail of 24 bases, there is the AATCAA sequence 55 nucleotides upstream which probably serves as a polyadenylation signal. However, AATCAA directs polyadenylation in vitro with very low efficiency. We found no open reading frame in the clone and this is in agreement with the data indicating that brain-specific RNAs has extremely long 3'-untranslated regions. Hmob3 was partially sequences. We compared its primary structure with the sequences from the Gene Bank and revealed no homology. Hmob3 expresses in different parts of human brain and in sceletal muscle but does not express in other tissues.

[Analysis of sequences from human brain cDNA gene bank which are functionally active in nervous tissue and tumor cells]. / Analiz posledovatel'nostei, poluchennykh iz banka kDNK mozga cheloveka i aktivno funktsioniruiushchikh v nervnykh i opukholevykh kletkakh.

Construction of a human cortex cDNA bank is described as well as the isolation from this bank of pBH71 and pBH3 clones with preferential expression in nervous and in tumor cells. The clones can be included into the third class of cDNA according to Sutcliff's classification. The mRNA corresponding to this cDNA class is considered to play the key role in determination of specificity of nervous tissue. Expression of the pBH71 sequence was revealed in human cortex and in tissues of different genesis (from neuroblastoma to uterus myoma), a 2 kb mRNA which corresponds to one and the same cDNA chain having been found in all tissues under analysis. The nucleotide sequence of cDNA insertion into the pBH71 clone of 447 n.p. was determined, and particular features of cDNA nucleotide composition and possible schemes of its translation were analysed. Weak homology was found between the 3'-end of cDNA insertion of the pBH71 clone and the 3'-end region of human proopiomelanocortine. The cDNA of the pBH3 clone hybridizes with the 0.8 kb mRNA revealed in human cortex and neuroendocrine tumors of different nature. No homology was revealed between the cDNA sequence of the pBH3 clone and any genes deciphered.