Fas antigen is a cell-surface protein that mediates apoptosis. It is expressed in various tissues including the thymus and has structural homology with a number of cell-surface receptors, including tumour necrosis factor receptor and nerve growth factor receptor. Mice carrying the lymphoproliferation (lpr) mutation have defects in the Fas antigen gene. The lpr mice develop lymphadenopathy and suffer from a systemic lupus erythematosus-like autoimmune disease, indicating an important role for Fas antigen in the negative selection of autoreactive T cells in the thymus.

The mouse H19 gene was identified as an abundant hepatic fetal-specific mRNA under the transcriptional control of a trans-acting locus termed raf. The For the majority of cloned eucaryotic genes, their protein products were already known prior to cloning. More recently, differential hybridization screens of cDNA libraries have been used to clone genes with specific tissue and temporal patterns of expression, without regard to their products. The murine H19 gene is such a gene. It was originally identified in a screen of a fetal liver cDNA library to find genes that were coordinately regulated with the a-fetoprotein (AFP) gene (25). The aim of the screen was to find fetal-specific mRNAs under the control of raf, a transacting locus that determines, at least in part, the adult basal level of AFP mRNA (2, 24). The only cDNA that fulfilled these criteria was the H19 cDNA. This gene was expressed at high levels in fetal and neonatal liver, and its 600-fold repression in the adult was under the control of raf. Its coordinate expression with AFP extended to the visceral endoderm of the yolk sac and the fetal gut. However, unlike AFP, H19 mRNA was also found in both fetal skeletal and cardiac muscle at high levels and at reduced levels in all adult muscle, although in these tissues it is not under the influence of raf (2).Sequence analysis of the single-copy H19 gene revealed the presence of multiple small open reading frames (ORFs), none of which spanned more than two of its five exons. The largest of these, called ORF5 (referred to as ORF1 in reference 26), was located entirely within the first exon and could potentially encode a 132-amino-acid protein. This ORF begins 680 bases downstream of the cap site and is preceded by four small ORFs, the largest of which could encode a protein of 27 amino acids. As there was no precedent for such a gene organization, we decided to investigate its possible significance.As a first step, we cloned and sequenced the H19 gene from the human genome, reasoning that a comparison between the murine and human homologs would identify regions that are conserved and therefore important to the function of the gene. The demonstration that the genes shared no ORF led us to investigate the subcellular distribution of H19 mRNA, which indicated that the majority of the mRNA is located in a cytoplasmic particle. * Corresponding author. MATERIALS AND METHODScDNA library construction. A 20-,Ig sample of poly(A)+ RNA from Hep3B human hepatoma cells in 90 ,l of distilled H20 was heat denatured at 65°C for 3 min and then placed at 4°C. To this was added 20 ,ug of oligo(dT) (Boehringer Mannheim Biochemicals), 80 U of RNasin (Promega Biotec), 1.5 mM each dGTP, dATP, dCTP, and dTTP, 50 mM Tris hydrochloride (pH 8.3), 10 mM MgCl2, 70 mM KCI, 10 mM dithiothreitol, and 300 U of reverse transcriptase (Life Sciences, Inc.) to a final reaction volume of 200 ,ul; the mixture was incubated at 42°C for 1 h. This preparation was then mixed with 400 ,u1 of a solution containing 20 mM Tris hydrochloride (pH 7.5), 5...

We have identified three C͞D-box small nucleolar RNAs (snoRNAs) and one H͞ACA-box snoRNA in mouse and human. In mice, all four snoRNAs (MBII-13, MBII-52, MBII-85, and MBI-36) are exclusively expressed in the brain, unlike all other known snoRNAs. Two of the human RNA orthologues (HBII-52 and HBI-36) share this expression pattern, and the remainder, HBII-13 and HBII-85, are prevalently expressed in that tissue. In mice and humans, the brain-specific H͞ACA box snoRNA (MBI-36 and HBI-36, respectively) is intronencoded in the brain-specific serotonin 2C receptor gene. The three human C͞D box snoRNAs map to chromosome 15q11-q13, within a region implicated in the Prader-Willi syndrome (PWS), which is a neurogenetic disease resulting from a deficiency of paternal gene expression. Unlike other C͞D box snoRNAs, two snoRNAs, HBII-52 and HBII-85, are encoded in a tandemly repeated array of 47 or 24 units, respectively. In mouse the homologue of HBII-52 is processed from intronic portions of the tandem repeats. Interestingly, these snoRNAs were absent from the cortex of a patient with PWS and from a PWS mouse model, demonstrating their paternal imprinting status and pointing to their potential role in the etiology of PWS. Despite displaying hallmarks of the two families of ubiquitous snoRNAs that guide 2-O-ribose methylation and pseudouridylation of rRNA, respectively, they lack any telltale rRNA complementarity. Instead, brain-specific C͞D box snoRNA HBII-52 has an 18-nt phylogenetically conserved complementarity to a critical segment of serotonin 2C receptor mRNA, pointing to a potential role in the processing of this mRNA. T he biogenesis of eukaryotic ribosomes involves a complex rRNA processing pathway mostly taking place in a specialized subnuclear compartment, the nucleolus. Pre-rRNA maturation includes, in addition to a series of endonucleolytic and exonucleolytic cleavages, the covalent modification of a definite subset of rRNA nucleotides, essentially by 2Ј-O-ribose methylation and pseudouridylation. Each of these modifications is found at about 100 sites per vertebrate ribosome (1). Although these modifications are phylogenetically conserved and restricted to the most highly conserved and functionally important regions of rRNA, their function remains largely unknown. Spliceosomal small nuclear RNAs (snRNAs) also contain a number of conserved 2Ј-Omethylated nucleotides and pseudouridines, confined to snRNA sequences critical for splicing, i.e., involved in contacts with premRNA or other snRNAs (2). Remarkably, nucleotide modifications in the 5Ј terminal region of U2 snRNA are required for assembly of a functional U2 sn-ribonucleoprotein particle (3).The nucleolus contains a large number of small, metabolically stable RNAs, termed small nucleolar RNAs (snoRNAs) that fall into two major classes, the C͞D box and H͞ACA box snoRNAs, designated after common sequence motifs involved in the assembly of sno-ribonucleoprotein particles. Although each class includes a small number of snoRNAs required for definite pre-rRNA cl...

The neurofibromatosis {NF1) gene shows significant homology to mammalian GAP and is an important regulator of the ras signal transduction pathway. To study the function of NF1 in normal development and to try and develop a mouse model of NF1 disease, we have used gene targeting in ES cells to generate mice carrying a null mutation at the mouse Nfl locus. Although heterozygous mutant mice, aged up to 10 months, have not exhibited any obvious abnormalities, homozygous mutant embryos die in utero. Embryonic death is likely attributable to a severe malformation of the heart. Interestingly, mutant embryos also display hyperplasia of neural crest-derived sympathetic ganglia. These results identify new roles for NF1 in development and indicate that some of the abnormal growth phenomena observed in NF1 patients can be recapitulated in neurofibromin-deficient mice.