Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.

The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins.

Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region.

The Tsc2 gene, which is mutationally inactivated in the germ line of some families with tuberous sclerosis, encodes a large, membrane-associated GTPase activating protein (GAP) designated tuberin. Studies of the Eker rat model of hereditary cancer strongly support the role of Tsc2 as a tumor suppressor gene. In this study, the biological activity of tuberin was assessed by expressing the wild-type Tsc2 gene in tumor cell lines lacking functional tuberin and also in rat fibroblasts with normal levels of endogenous tuberin. The colony forming efficiency of Eker rat-derived renal carcinoma cells was significantly reduced following reintroduction of wild-type Tsc2. Tumor cells expressing the transfected Tsc2 gene became more anchorage-dependent and lost their ability to form tumors in severe combined immunodeficient mice. At the cellular level, restoration of tuberin expression caused morphological changes characterized by enlargement of the cells and increased contact inhibition. As with the full-length Tsc2 gene, a clone encoding only the C terminus of tuberin (amino acids 1049-1809, including the GAP domain) was capable of reducing both colony formation and in vivo tumorigenicity when transfected into the Eker rat tumor cells. In normal Rat1 fibroblasts, conditional overexpression of tuberin also suppressed colony formation and cell growth in vitro. These results provide direct experimental evidence for the tumor suppressor function of Tsc2 and suggest that the tuberin C terminus plays an important role in this activity.

Cloning and characterization of KAP3: a novel kinesin superfamily-associated protein of KIF3A/3B.

We previously reported that KIF3A and KIF3B form a heterodimer that functions as a microtubule-based fast anterograde translocator of membranous organelles. We have also shown that this KIF3A/3B forms a complex with other associated polypeptides, named kinesin superfamily-associated protein 3 (KAP3). In the present study, we purified KAP3 protein by immunoprecipitation using anti-KIF3B antibody from mouse testis. Microsequencing was carried out, and we cloned the full-length KAP3 cDNA from a mouse brain cDNA library. Two isoforms of KAP3 exist [KAP3A (793 aa) and KAP3B (772 aa)], generated by alternative splicing in the carboxyl terminus region. Their amino acid sequences have no homology with those of any other known proteins, and prediction of their secondary structure indicated that almost the entire KAP3 molecule is alpha-helical. We produced recombinant KAP3 and KIF3A/3B using a baculovirus-Sf9 expression system. A reconstruction study in Sf9 cells revealed that KAP3 is a globular protein that binds to the tail domain of KIF3A/3B. The immunolocalization pattern of KAP3 was similar to that of KIF3A/3B in nerve cells. In addition, we found that KAP3 does not affect the motor activity of KIF3A/3B. KAP3 was associated with a membrane-bound form of KIF3A/3B in a fractional immunoprecipitation experiment, and since the KIF3 complex was found to bind to membranous organelles in an EM study, KAP3 may regulate membrane binding of the KIF3 complex.

Transgenic expression of PML/RARalpha impairs myelopoiesis.

The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene (PML) on chromosome 15 with the retinoic acid receptor alpha (RARalpha) on chromosome 17. This yields a fusion transcript, PML/RARalpha, a transcription factor with reported dominant negative functions in the absence of hormone. Clinical remissions induced with all-trans retinoic acid (RA) treatment in acute promyelocytic leukemia are linked to PML/RARalpha expression in leukemic cells. To evaluate the PML/RARalpha role in myelopoiesis, transgenic mice expressing PML/RARalpha were engineered. A full-length PML/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice. Expression was confirmed in the bone marrow with a reverse transcription PCR assay. Basal total white blood cell and granulocyte counts did not appreciably differ between PML/RARalpha transgenic and control mice. Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice. However, in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors, especially in those responding to granulocyte/ macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition. Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation. Following irradiation, PML/RARalpha transgenic mice, as compared with controls, more rapidly depressed peripheral white blood cell and granulocyte counts. As expected, nearly all control mice (94.4%) survived irradiation, yet this irradiation was lethal to 45.8% of PML/RARalpha transgenic mice. Lethality was associated with more severe leukopenia in transgenic versus control mice. Retinoic acid treatment of irradiated PML/RARalpha mice enhanced granulocyte recovery. These data suggest that abnormal myelopoiesis due to PML/RARalpha expression is an early event in oncogenic transformation.

Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis.

The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA. Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region. The processed RNA is significantly more stable than the full-length transcript. Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript. However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form. This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions. The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination. Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.

Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism.

RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport.

Highly stable expression of a foreign gene from rabies virus vectors.

A reverse genetics approach was applied to generate a chimeric nonsegmented negative strand RNA virus, rabies virus (RV) of the Rhabdoviridae family, that expresses a foreign protein. DNA constructs containing the entire open reading frame of the bacterial chloramphenicol acetyltransferase (CAT) gene and an upstream RV cistron border sequence were inserted either into the nontranslated pseudogene region of a full-length cDNA copy of the RV genome or exchanged with the pseudogene region. After intracellular T7 RNA polymerase-driven expression of full-length antigenome RNA transcripts and RV nucleoprotein, phosphoprotein and polymerase from transfected plasmids, RVs transcribing novel monocistronic mRNAs and expressing CAT at high levels, were recovered. The chimeric viruses possessed the growth characteristics of standard RV and were genetically stable upon serial cell culture passages. CAT activity was still observed in cell cultures infected with viruses passaged for more than 25 times. Based on the unprecedented stability of the chimeric RNA genomes, which is most likely due to the structure of the rhabdoviral ribonucleoprotein complex, we predict the successful future use of recombinant rhabdovirus vectors for displaying foreign antigens or delivering therapeutic genes.

IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus.

IA-2 is a 105,847 Da transmembrane protein that belongs to the protein tyrosine phosphatase family. Immunoperoxidase staining with antibody raised against IA-2 showed that this protein is expressed in human pancreatic islet cells. In this study, we expressed the full-length cDNA clone of IA-2 in a rabbit reticulocyte transcription/translation system and used the recombinant radiolabeled IA-2 protein to detect autoantibodies by immunoprecipitation. Coded sera (100) were tested: 50 from patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) and 50 from age-matched normal controls. Sixty-six percent of the sera from patients, but none of the sera from controls, reacted with IA-2. The same diabetic sera tested for autoantibodies to islet cells (ICA) by indirect immunofluorescence and glutamic acid decarboxylase (GAD65Ab) by depletion ELISA showed 68% and 52% positivity, respectively. Up to 86% of the IDDM patients had autoantibodies to IA-2 and/or GAD65. Moreover, greater than 90% (14 of 15) of the ICA-positive but GAD65Ab-negative sera had autoantibodies to IA-2. Absorption experiments showed that the immunofluorescence reactivity of ICA-positive sera was greatly reduced by prior incubation with recombinant IA-2 or GAD65 when the respective antibody was present. A little over one-half (9 of 16) of the IDDM sera that were negative for ICA were found to be positive for autoantibodies to IA-2 and/or GAD65, arguing that the immunofluorescence test for ICA is less sensitive than the recombinant tests for autoantibodies to IA-2 and GAD65. It is concluded that IA-2 is a major islet cell autoantigen in IDDM, and, together with GAD65, is responsible for much of the reactivity of ICA with pancreatic islets. Tests for the detection of autoantibodies to recombinant IA-2 and GAD65 may eventually replace ICA immunofluorescence for IDDM population screening.

In vitro motility from recombinant dynein heavy chain.

The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain.

Molecular cloning of the first metazoan beta-1,3 glucanase from eggs of the sea urchin Strongylocentrotus purpuratus.

We report the molecular cloning of the first beta-1,3 glucanase from animal tissue. Three peptide sequences were obtained from beta-1,3 glucanase that had been purified from eggs of the sea urchin Strongylocentrotus purpuratus and the gene was cloned by PCR using oligonucleotides deduced from the peptide sequences. The full-length cDNA shows a predicted enzyme structure of 499 aa with a hydrophobic signal sequence. A 3.2-kb message is present in eggs, during early embryogenesis, and in adult gut tissue. A polyclonal antibody to the native 68-kDa enzyme recognizes a single band during early embryogenesis that reappears in the adult gut, and recognizes a 57-kDa fusion protein made from a full-length cDNA clone for beta-1,3 glucanase. The identity of this molecule as beta-1,3 glucanase is confirmed by sequence homology, by the presence of all three peptide sequences in the deduced amino acid sequence, and by the recognition of the bacterial fusion protein by the antibody directed against the native enzyme. Data base searches show significant homology at the amino acid level to beta-1,3 glucanases from two species of bacteria and a clotting factor from the horseshoe crab. The homology with the bacteria is centered in a 304-aa region in which there are seven scattered regions of high homology between the four divergent species. These four species were also found to have two homologous regions in common with more distantly related plant, fungal, and bacterial proteins. A global phylogeny based on these regions strongly suggests that the glucanases are a very ancient family of genes. In particular, there is an especially deep split within genes taken from the bacterial genus Bacillus.

Cloning of rat MEK kinase 1 cDNA reveals an endogenous membrane-associated 195-kDa protein with a large regulatory domain.

The coding sequence of rat MEK kinase 1 (MEKK1) has been determined from multiple, independent cDNA clones. The cDNA is full-length based on the presence of stop codons in all three reading frames of the 5' untranslated region. Probes from the 5' and the 3' coding sequences both hybridize to a 7-kb mRNA. The open reading frame is 4.5 kb and predicts a protein with molecular mass of 161,225 Da, which is twice the size of the previously published MEKK1 sequence and reveals 801 amino acids of novel coding sequence. The novel sequence contains two putative pH domains, two proline-rich regions, and a cysteine-rich region. Antisera to peptides derived from this new sequence recognize an endogenous protein in human and rodent cells of 195 kDa, consistent with the size of the expressed rat MEKK1 clone. Endogenous and recombinant rat MEKK1 are enriched in membranes; little of either is found in soluble fractions. Expression of recombinant rat MEKK1 leads to activation of three mitogen-activated protein kinase modules in the order c-Jun N-terminal kinase/stress-activated protein kinase > p38 mitogen-activated protein kinase = extracellular signal-regulated kinase 2.

The transcription factors c-myb and GATA-2 act independently in the regulation of normal hematopoiesis.

The transcription factors c-myb and GATA-2 are both required for blood cell development in vivo and in vitro. However, very little is known on their mechanism(s) of action and whether they impact on complementary or overlapping pathways of hematopoietic proliferation and differentiation. We report here that embryonic stem (ES) cells transfected with c-myb or GATA-2 cDNAs, individually or in combination, underwent hematopoietic commitment and differentiation in the absence of added hematopoietic growth factors but that stimulation with c-kit and flt-3 ligands enhanced colony formation only in the c-myb transfectants. This enhancement correlated with c-kit and flt-3 surface receptor up-regulation in c-myb-(but not GATA-2-) transfected ES cells. Transfection of ES cells with either a c-myb or a GATA-2 antisense construct abrogated erythromyeloid colony-forming ability in methyl cellulose; however, introduction of a full-length GATA-2 or c-myb cDNA, respectively, rescued the hematopoiesis-deficient phenotype, although only c-myb-rescued ES cells expressed c-kit and flt-3 surface receptors and formed increased numbers of hematopoietic colonies upon stimulation with the cognate ligands. These results are in agreement with previous studies indicating a fundamental role of c-myb and GATA-2 in hematopoiesis. Of greater importance, our studies suggest that GATA-2 and c-myb exert their roles in hematopoietic gene regulation through distinct mechanisms of action in nonoverlapping pathways.

Mechanism for transcriptional gain of function resulting from chromosomal translocation in alveolar rhabdomyosarcoma.

The t(2;13) translocation of alveolar rhabdomyosarcoma results in tumor-specific expression of a chimeric transcription factor containing the N-terminal DNA-binding domain of PAX3 and the C-terminal transactivation domain of FKHR. Here we have tested the hypothesis that PAX3-FKHR gains function relative to PAX3 as a consequence of switching PAX3 and FKHR transactivation domains, which were previously shown to have similar potency but distinct structural motifs. In transient cotransfection assays with human expression constructs, we have demonstrated the increased ability of PAX3-FKHR to activate transcription of a reporter gene located downstream of multimerized e5, PRS-9, or CD19 DNA-binding sites in three cell lines. For example, PAX3-FKHR was 100-fold more potent than PAX3 as an activator binding to e5 sites in NIH 3T3 cells. To compare transactivation potency independent of PAX3-specific DNA binding, we tested GAL4 fusions of full-length PAX3 and PAX3-FKHR or their respective C-terminal transactivation domains on a reporter with GAL4 DNA-binding sites. In this context, full-length PAX3-FKHR was also much more potent than PAX3. Additionally, the activity of each full-length protein was decreased relative to its C-terminal domain, demonstrating that N-terminal sequences are inhibitory. By deletion analysis, we mapped a bipartite cis-acting inhibitory domain to the same subregions within the DNA-binding domains of both PAX3 and PAX3-FKHR. We have shown, however, that the structurally distinct transactivation domains of PAX3 and PAX3-FKHR differ 10- to 100-fold in their susceptibility to inhibition, thus elucidating a mechanism by which PAX3 gains enhanced function during oncogenesis.

Cloning and functional expression of cDNAs encoding human and rat pancreatic polypeptide receptors.

PCR was used to isolate nucleotide sequences that may encode novel members of the neuropeptide Y receptor family. By use of a PCR product as a hybridization probe, a full-length human cDNA was isolated that encodes a 375-aa protein with a predicted membrane topology identifying it as a member of the G-protein-coupled receptor superfamily. After stable transfection of the cDNA into human embryonic kidney 293 cells, the receptor exhibited high affinity (Kd = 2.8 nM) for 125I-labeled human pancreatic polypeptide (PP). Competition binding studies in whole cells indicated the following rank order of potency: human PP = bovine PP > or = human [Pro34]peptide YY > rat PP > human peptide YY = human neuropeptide Y. Northern blot analysis revealed that human PP receptor mRNA is most abundantly expressed in skeletal muscle and, to a lesser extent, in lung and brain tissue. A rat cDNA clone encoding a high-affinity PP receptor that is 74% identical to the human PP receptor at the amino acid level was also isolated. These receptor clones will be useful in elucidating the functional role of PP and designing selective PP receptor agonists and antagonists.

Molecularly engineered resistance to California serogroup virus replication in mosquito cells and mosquitoes.

Introduction of genetic elements derived from a viral pathogen's genome may be used to reduce the vectorial capacity of mosquitoes for that virus. A double subgenomic Sindbis virus expression system was utilized to transcribe sequences of LaCrosse (LAC) virus small (S) or medium (M) segment RNA in sense or antisense orientation; wild-type Sindbis and LaCrosse viruses have single-stranded RNA genomes, the former being positive sense and the latter being negative sense. Recombinant viruses were generated and used to infect Aedes albopictus (C6/36) mosquito cells, which were challenged with wild-type LAC virus and then assayed for LAC virus replication. Several recombinant viruses containing portions of the LAC S segment were capable of inducing varying degrees of interference to the challenge virus. Cells infected with TE/3'2J/ANTI-S virus, expressing full-length negative-sense S RNA of LAC virus, yielded 3-6 log10TCID50 (tissue culture 50% infective dose) less LAC virus per ml than did cells infected with a double subgenomic sindbis virus containing no LAC insert. When C6/36 cells infected with TE/3'2J/ANTI-S were challenged with closely related heterologous bunyaviruses, a similar inhibitory effect was seen. Adult Ae. triseriatus mosquitoes infected with TE/3'2J/ANTI-S were also resistant to challenge by LAC virus. Organs that were productively infected by the double subgenomic Sindbis virus expressing the LAC anti-S sequences demonstrated little LAC virus or antigen. These studies indicate that expression of carefully selected antiviral sequences derived from the pathogen's genome may result in efficacious molecular viral interference in mosquito cells and, more importantly, in mosquitoes.