A targeted mutation of the D3 dopamine receptor gene is associated with hyperactivity in mice.

While most effects of dopamine in the brain are mediated by the D1 and D2 receptor subtypes, other members of this G protein-coupled receptor family have potentially important functions. D3 receptors belong to the D2-like subclass of dopamine receptors, activation of which inhibits adenylyl cyclase. Using targeted mutagenesis in mouse embryonic stem cells, we have generated mice lacking functional D3 receptors. A premature chain-termination mutation was introduced in the D3 receptor gene after residue Arg-148 in the second intracellular loop of the predicted protein sequence. Binding of the dopamine antagonist [125I]iodosulpride to D3 receptors was absent in mice homozygous for the mutation and greatly reduced in heterozygous mice. Behavioral analysis of mutant mice showed that this mutation is associated with hyperactivity in an exploratory test. Homozygous mice lacking D3 receptors display increased locomotor activity and rearing behavior. Mice heterozygous for the D3 receptor mutation show similar, albeit less pronounced, behavioral alterations. Our findings indicate that D3 receptors play an inhibitory role in the control of certain behaviors.

Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.

An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins.

Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode surface coat, which is shed in response to binding by host antibody molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein series. We have isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of TES-120. The mRNA is absent from T. canis adults but hyperabundant in larvae, making up approximately 10% of total mRNA, and is trans-spliced with the nematode 5' leader sequence SL1. It encodes a 15.8-kDa protein (after signal peptide removal) containing a typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized into an array of heptameric repeats, interspersed with proline residues. At the C-terminal end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a motif that can also be identified in several genes in Caenorhabditis elegans. Although TES-120 displays size and charge heterogeneity, there is a single copy gene and a homogeneous size of mRNA. The association of overexpression of some membrane-associated mucins with immunosuppression and tumor metastasis suggests a possible model for the role of the surface coat in immune evasion by parasitic nematodes.

An X chromosome-linked gene encoding a protein with characteristics of a rhoGAP predominantly expressed in hematopoietic cells.

An increasingly large number of proteins involved in signal transduction have been identified in recent years and shown to control different steps of cell survival, proliferation, and differentiation. Among the genes recently identified at the tip of the long arm of the human X chromosome, a novel gene, C1, encodes a protein that appears to represent a newly discovered member of the group of signaling proteins involved in regulation of the small GTP binding proteins of the ras superfamily. The protein encoded by C1, p115, is synthesized predominantly in cells of hematopoietic origin. It is characterized by two regions of similarity to motifs present in known proteins: GAP and SH3 homologous regions. Its localization in a narrow cytoplasmic region just below the plasma membrane and its inhibitory effect on stress fiber organization indicate that p115 may down regulate rho-like GTPases in hematopoietic cells.

Tissue-specific response of the human platelet-activating factor receptor gene to retinoic acid and thyroid hormone by alternative promoter usage.

We have studied the effects of retinoic acid (RA) and thyroid hormone (3,3',5-triiodothyronine; T3) on platelet-activating factor receptor (PAFR) gene expression in intact rats and the ability of two human PAFR gene promoters (PAFR promoters 1 and 2) to generate two transcripts (PAFR transcripts 1 and 2). Northern blotting showed that RA and T3 regulated PAFR gene expression only in rat tissues that express PAFR transcript 2. Functional analysis of the human PAFR promoter 2 revealed that responsiveness to RA and T3 was conferred through a 24-bp element [PAFR-hormone response element (HRE) located from -67 to -44 bp of the transcription start site, whereas PAFR promoter 1 did not respond to these hormones. The PAFR-HRE is composed of three direct repeated TGACCT-like hexamer motifs with 2-and 4-bp spaces, and the two upstream and two downstream motifs were identified as response elements for RA and T3. Thus, the PAF-PAFR pathway is regulated by the PAFR level altered by a tissue-specific response to RA and T3 through the PAFR-HRE of the PAFR promoter 2.

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.

The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression.

The retinal protein Nrl belongs to a distinct subfamily of basic motif-leucine zipper DNA-binding proteins and has been shown to bind extended AP-1-like sequence elements as a homo- or heterodimer. Here, we demonstrate that Nrl can positively regulate the expression of the photoreceptor cell-specific gene rhodopsin. Electrophoretic mobility-shift analysis reveals that a protein(s) in nuclear extracts from bovine retina and the Y79 human retinoblastoma cell line binds to a conserved Nrl response element (NRE) in the upstream promoter region of the rhodopsin gene. Nrl or an antigenically similar protein is shown to be part of the bound protein complex by supershift experiments using Nrl-specific antiserum. Cotransfection studies using an Nrl-expression plasmid and a luciferase reporter gene demonstrate that interaction of the Nrl protein with the -61 to -84 region of the rhodopsin promoter (which includes the NRE) stimulates expression of the reporter gene in CV-1 monkey kidney cells. This Nrl-mediated transactivation is specifically inhibited by coexpression of a naturally occurring truncated form of Nrl (dominant negative effect). Involvement of Nrl in photoreceptor gene regulation and its continued high levels of expression in the adult retina suggest that Nrl plays a significant role in controlling retinal function.

Cloning and comparative analysis of the human pre-T-cell receptor alpha-chain gene.

In immature T cells the T-cell receptor (TCR) beta-chain gene is rearranged and expressed before the TCR alpha-chain gene. At this stage TCR beta chain can form disulfide-linked heterodimers with the pre-T-cell receptor alpha chain (pTalpha). Using the recently isolated murine pTalpha cDNA as a probe, we have isolated the human pTalpha cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTalpha with the mouse pTalpha molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTalpha gene is expressed in immature but not mature T cells and is located at the p21.2-p12 region of the short arm of chromosome 6.

Identification and expression analysis of a potential familial Alzheimer disease gene on chromosome 1 related to AD3.

The inheritance of much early-onset Alzheimer disease (AD) has been linked to a dominant-acting locus on chromosome 14. Recently, the gene likely responsible for this genetic linkage has been identified and termed AD3. Five mutations have been found in AD3 that segregate with the disease phenotype in seven AD families and are not present in unaffected individuals. Here we report the existence of a gene encoding a seven transmembrane domain protein very similar to that encoded by AD3 in structure and sequence. This gene is located on chromosome 1, is expressed in a variety of tissues, including brain, and is predicted to harbor mutations causing nonchromosome 14 familial AD. The presence of several S/TPXX DNA binding motifs in both the AD3 protein and the AD3-like protein /AD4 protein suggests a possible role in intracellular signaling and gene expression or in linking chromatin to the nuclear membrane. Ways in which mutations in either gene could lead to AD are discussed.

Role of insulin-like growth factors and myogenin in the altered program of proliferation and differentiation in the NFB4 mutant muscle cell line.

In the present study we used the mutant muscle cell line NFB4 to study the balance between proliferation and myogenic differentiation. We show that removal of serum, which induced the parental C2C12 cells to withdraw from the cell cycle and differentiate, had little effect on NFB4 cells. Gene products characteristic of the proliferation state, such as c-Jun, continued to accumulate in the mutant cells in low serum, whereas those involved in differentiation, like myogenin, insulin-like growth factor II (IGF-II), and IGF-binding protein 5 (IGFBP-5) were undetectable. Moreover, NFB4 cells displayed a unique pattern of tyrosine phosphorylated proteins, especially in low serum, suggesting that the signal transduction pathway(s) that controls differentiation is not properly regulated in these cells. Treatment of NFB4 cells with exogenous IGF-I or IGF-II at concentrations shown to promote myogenic differentiation in wild-type cells resulted in activation of myogenin but not MyoD gene expression, secretion of IG-FBP-5, changes in tyrosine phosphorylation, and enhanced myogenic differentiation. Similarly, transfection of myogenin expression constructs also enhanced differentiation and resulted in activation of IGF-II expression, showing that myogenin and IGF-II cross-activate each other's expression. However, in both cases, the expression of Jun mRNA remained elevated, suggesting that IGFs and myogenin cannot overcome all aspects of the block to differentiation in NFB4 cells.

The gene distribution of the maize genome.

Previous investigations from our laboratory showed that the genomes of plants, like those of vertebrates, are mosaics of isochores, i.e., of very long DNA segments that are compositionally homogeneous and that can be subdivided into a small number of families characterized by different GC levels (GC is the mole fraction of guanine+cytosine). Compositional DNA fractions corresponding to different isochore families were used to investigate, by hybridization with appropriate probes, the gene distribution in vertebrate genomes. Here we report such a study on the genome of a plant, maize. The gene distribution that we found is most striking, in that almost all genes are present in isochores covering an extremely narrow (1-2%) GC range and only representing 10-20% of the genome. This gene distribution, which seems to characterize other Gramineae as well, is remarkably different from the gene distribution previously found in vertebrate genomes.

Fasciclin II controls proneural gene expression in Drosophila.

Fasciclin II (Fas II), an NCAM-like cell adhesion molecule in Drosophila, is expressed on a subset of embryonic axons and controls selective axon fasiculation. Fas II is also expressed in imaginal discs. Here we use genetic analysis to show that Fas II is required for the control of proneural gene expression. Clusters of cells in the eye-antennal imaginal disc express the achaete proneural gene and give rise to mechanosensory neurons; other clusters of cells express the atonal gene and give rise to ocellar photoreceptor neurons. In fasII loss-of-function mutants, the expression of both proneural genes is absent in certain locations, and, as a result, the corresponding sensory precursors fail to develop. In fasII gain-of-function conditions, extra sensory structures arise from this same region of the imaginal disc. Mutations in the Abelson tyrosine kinase gene show dominant interactions with fasII mutations, suggesting that Abl and Fas II function in a signaling pathway that controls proneural gene expression.

Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor.

Transcription factor CREM (cAMP-responsive element modulator) plays a pivotal role in the nuclear response to cAMP in neuroendocrine cells. We have previously shown that follicle-stimulating hormone (FSH) directs CREM expression in male germ cells. The physiological importance of FSH in Sertoli cell function prompted us to analyze its effect on CREM expression in these cells. We observed a dramatic and specific increase in the CREM isoform ICER (inducible cAMP early repressor) expression, with a peak 4 h after FSH treatment of primary Sertoli cells. Interestingly, induced levels of ICER protein persist for a considerably longer time. Induction of the repressor ICER accompanies early down-regulation of the FSH receptor transcript, which leads to long-term desensitization. Here we show that ICER represses FSH receptor expression by binding to a CRE-like sequence in the regulatory region of the gene. Our results confirm the crucial role played by CREM in hormonal control and suggest its role in the long-term desensitization phenomenon of peptide membrane receptors.

Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts.

The mechanisms by which insulin-like growth factors (IGFs) can be both mitogenic and differentiation-promoting in skeletal myoblasts are unclear because these two processes are believed to be mutually exclusive in this tissue. The phosphorylation state of the ubiquitous nuclear retinoblastoma protein (Rb) plays an important role in determining whether myoblasts proliferate or differentiate: Phosphorylated Rb promotes mitogenesis, whereas un- (or hypo-) phosphorylated Rb promotes cell cycle exit and differentiation. We hypothesized that IGFs might affect the fate of myoblasts by regulating the phosphorylation of Rb. Although long-term IGF treatment is known to stimulate differentiation, we find that IGFs act initially to inhibit differentiation and are exclusively mitogenic. These early effects of IGFs are associated with maintenance of Rb phosphorylation typical of proliferating cells; upregulation of the gene expression of cyclin-dependent kinase 4 and cyclin D1, components of a holoenzyme that plays a principal role in mediating Rb phosphorylation; and marked inhibition of the gene expression of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that is essential in muscle differentiation. We also find that IGF-induced inhibition of differentiation occurs through a process that is independent of its mitogenic effects. We demonstrate, thus, that IGFs regulate Rb phosphorylation and cyclin D1 and cyclin-dependent kinase 4 gene expression; together with their biphasic effects on myogenin expression, these results suggest a mechanism by which IGFs are initially mitogenic and subsequently differentiation-promoting in skeletal muscle.

Thyroid-stimulating hormone (TSH)-directed induction of the CREM gene in the thyroid gland participates in the long-term desensitization of the TSH receptor.

Thyroid gland function is regulated by the hypothalamic-pituitary axis via the secretion of TSH, according to environmental, developmental, and circadian stimuli. TSH modulates both the secretion of thyroid hormone and gland trophism through interaction with a specific guanine nucleotide-binding protein-coupled receptor (TSH receptor; TSH-R), which elicits the activation of the cAMP-dependent signaling pathway. After TSH stimulation, the levels of TSH-R RNA are known to decrease dramatically within a few hours. This phenomenon ultimately leads to homologous long-term desensitization of the TSH-R. Here we show that TSH drives the induction of the inducible cAMP early repressor (ICER) isoform of the cAMP response element (CRE) modulator gene both in rat thyroid gland and in the differentiated thyroid cell line FRTL-5. The kinetics of ICER protein induction mirrors the down-regulation of TSH-R mRNA. ICER binds to a CRE-like sequence in the TSH-R promoter and represses its expression. Thus, ICER induction by TSH in the thyroid gland represents a paradigm of the molecular mechanism by which pituitary hormones elicit homologous long-term desensitization.