J Biol Chem, 1994 Sep 23, 269(38), 23722 - 30 Differential phosphorylation of neuronal substrates by catalytic subunits of Aplysia cAMP-dependent protein kinase with alternative N termini; Panchal RG et al.; cAMP-dependent protein kinase (PKA) is an important participant in neuronal modulation: the ability of neurons to change their properties in response to external stimuli . In Aplysia mechanosensory neurons, PKA plays roles in both short and long term presynaptic facilitation, which is a simple model for learning and memory . PKA in Aplysia is a collection of structurally and functionally diverse regulatory and catalytic (C) subunits . We have argued that this diversity may in part account for the ability of the enzyme to take part in neuronal events that are spatially and temporally separated . Here, we add credence to this hypothesis by showing that C subunits of Aplysia PKA with alternative N termini target different substrates in subcellular fractions from Aplysia neurons, despite their similar actions on synthetic peptide substrates . Purified recombinant CAPL-AN1A1, which has an N terminus that is homologous to the myristylated sequence described in mammals, catalyzes the formation of two phosphoproteins of 24 and 8 kDa more rapidly than CAPL-AN2A1, which has a distinct N terminus weakly related to that of the yeast TPK1 gene product . The 24-kDa phospoprotein, but not the 8-kDa species, is detected in taxol-stabilized microtubules, suggesting that it is associated with the cytoskeleton . CAPL-AN2A1, in contrast, generates a 55-kDa phosphoprotein that is not observed with CAPL-AN1A1 . The 55-kDa species is found in the detergent supernatant of the cytoskeleton fraction . Differential targeting of substrates by C subunits of PKA may therefore contribute to the ability of this kinase to play multiple roles in neuronal modulation. J Biol Chem, 1994 Sep 23, 269(38), 23590 - 6 Identification of exon sequences involved in splice site selection; Dominski Z et al.; The involvement of exon sequences in splice site selection was studied in vivo in HeLa cells transfected with a series of model three exon-two intron pre-mRNAs which differed only in the sequence of their internal exons . When the majority of the human globin-derived 175-nucleotide internal exon (DUP175) was replaced with a sequence from the yeast URA3 gene (DUP184), the splicing pathway changed from complete inclusion of the internal exon in DUP175 to its predominant skipping in the DUP184 construct . Skipping of the exon was reversed by increasing the strength of its flanking splicing elements indicating that exon sequences exert their effect only in the presence of relatively weak splicing signals . A series of block mutations in the internal exon of DUP184 showed that a stretch of 6 cytidine nucleotides increased the inclusion of the DUP184 internal exon about 7-fold . Mutations generating purine-rich sequences (AAG and GAAG) at the 3' end of the exon led to complete exon inclusion while stepwise insertion of sequences from the internal exon of DUP175 into the DUP184 background increased exon inclusion 5-fold . Combination of the stretch of cytidines with sequences derived from DUP175 exon resulted in complete exon inclusion indicating that diverse signals within exons may cooperate with each other in affecting splice site selection. Eur J Pharmacol, 1994 Sep 22, 263(1-2), 81 - 4 Fluoxetine reduces inflammatory edema in the rat: involvement of the pituitary-adrenal axis; Bianchi M et al.; The acute effect of the non-tricyclic, pro-serotoninergic, antidepressant drug fluoxetine on inflammatory edema was evaluated in the rat . Fluoxetine significantly and dose dependently reduced the swelling induced by the injection of 10% brewer's yeast suspension in the hindpaw . Both adrenalectomy and hypophysectomy prevented the effect of fluoxetine . In contrast pretreatment with the corticotropin-releasing hormone antagonist alpha-helical CRH-(9-41) did not interfere with the anti-inflammatory action of fluoxetine . Moreover, the drug induced a significant increase of corticosterone plasma concentrations in vivo, whereas, in vitro, it did not stimulate beta-endorphin release from anterior pituitary cells . Our data suggest that fluoxetine exerts a potent anti-inflammatory action by inducing pituitary-adrenocortical activation via serotonin. Nature, 1994 Sep 22, 371(6495), 355 - 8 Calcium signalling in T cells stimulated by a cyclophilin B-binding protein; Bram RJ et al.; The immunosuppressant drug cyclosporin A blocks a calcium-dependent signal from the T-cell receptor (TCR) that normally leads to T-cell activation . When bound to cyclophilin, cyclosporin A binds and inactivates the key signalling intermediate calcineurin . To identify potential cellular homologues of cyclosporin A that might regulate calcium signalling, we have cloned human genes encoding cyclophilin B-binding-proteins using the yeast two-hybrid system . One gene product, when overexpressed in Jurkat T cells, specifically induced transcription from the interleukin-2 enhancer, by activating the T-cell-specific transcription factors NF-AT and NF-IL2A . This protein, termed calcium-signal modulating cyclophilin ligand (CAML), acts downstream of the TCR and upstream of calcineurin by causing an influx of calcium . CAML appears to be a new participant in the calcium-signal transduction pathway, implicating cyclophilin B in calcium signalling, even in the absence of cyclosporin. J Biol Chem, 1994 Sep 16, 269(37), 23128 - 34 Cytosolic NADP(+)-dependent isocitrate dehydrogenase . Isolation of rat cDNA and study of tissue-specific and developmental expression of mRNA; Jennings GT et al.; Immunoscreening and DNA hybridization were used to isolate a 1.72-kilobase pair cDNA encoding cytosolic NADP(+)-dependent isocitrate dehydrogenase from a rat liver, lambda gt11 cDNA library . The identity of the cDNA was confirmed by comparison of the deduced amino acid sequence with sequences of peptides obtained from purified ovarian cytosolic isocitrate dehydrogenase . The 1.72-kilobase pair cDNA sequence translated into a protein of 414 amino acid residues with a molecular mass of 46,681 Da . The amino acid sequence contains a tripeptide (AKL) at the COOH terminus which represents a possible peroxisomal targeting sequence . The deduced amino acid sequence of the rat liver cytosolic isocitrate dehydrogenase showed 70 and 59% identity with sequences reported for NADP(+)-dependent isocitrate dehydrogenases from porcine mitochondria and yeast cytosol respectively . Northern blot analysis demonstrated a 13-fold increase in expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase mRNA during the gonadotropin-induced development of the immature rat ovary . In comparative studies, the cytosolic and mitochondrial isocitrate dehydrogenase mRNAs were found to differ in size (2.2 and 1.8 kilobases, respectively) and to be differentially expressed in various tissues of the rat . Distinct digestion patterns were also obtained in Southern blot analysis of rat genomic DNA. J Biol Chem, 1994 Sep 16, 269(37), 22925 - 8 Crkl is the major tyrosine-phosphorylated protein in neutrophils from patients with chronic myelogenous leukemia; Oda T et al.; The Philadelphia chromosome (Ph1), detected in virtually all cases of chronic myelogenous leukemia (CML), is formed by a reciprocal translocation between chromosome 9 and 22 that fuses Bcr-encoded sequences upstream of exon 2 of c-Abl . This oncogene produces a fusion protein, p210bcr-abl, in which the Abl tyrosine kinase activity is elevated . Using anti-phosphotyrosine immunoblotting, we have compared the pattern of phosphotyrosine-containing proteins from freshly prepared neutrophils of patients in the stable phase of CML to normal controls . The only consistent difference was the presence of a 39-kDa tyrosine-phosphorylated protein in 18 out of 18 neutrophil samples from CML patients that was not seen in normal controls . This same protein, as assessed by two-dimensional anti-phosphotyrosine immunoblotting, was also present in cell lines expressing p210bcr-abl, including K562 cells . Using K562 cells as a source of protein, the 39-kDa protein was purified and identified by microsequencing as Crkl, an SH2/SH3 adaptor protein related to the crk oncogene of the avian sarcoma virus, CT10 . A direct interaction between Crkl and Abl has also been shown using a yeast two-hybrid screen. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 1028 - 34 Differential decrease of copper content and of copper binding to superoxide dismutase in liver, heart and brain of copper-deficient rats; Rossi L et al.; Dietary copper-deficiency in rats produced a organ-specific decrease of copper content . This was paralleled by a decrease of the activity of the copper-enzyme superoxide dismutase . In liver such a decrease is partially due to the existence of an apo-form of superoxide dismutase, which can be reactivated by addition of exogenous copper to tissue extracts . These results demonstrate in vivo that superoxide dismutase is post-translationally modulated by copper in higher vertebrates as previously found for yeast and mammalian cells in culture. Blood, 1994 Sep 15, 84(6), 1747 - 52 ENL, the gene fused with HRX in t(11;19) leukemias, encodes a nuclear protein with transcriptional activation potential in lymphoid and myeloid cells; Rubnitz JE et al.; Chromosome band 11q23 is the site of recurring translocations with a variety of partner chromosomes in myeloid and lymphoid acute leukemias, infant leukemias, and treatment-induced secondary acute myelogenous leukemia . The translocation breakpoints cluster in a restricted region of the HRX gene resulting in fusion genes that encode an N-terminal portion of Hrx fused to various partner proteins . We have characterized the transcriptional transactivation properties of Enl, a protein that is fused to Hrx in t(11;19) leukemias . Enl is a nuclear protein that is capable of activating transcription from synthetic reporter genes in both lymphoid and myeloid cells, as well as in yeast . Deletion mutagenesis demonstrated that the minimal portion of Enl required for activation of transcription was localized to its C-terminal 90 amino acids . This region is highly conserved between Enl and the t(9;11) fusion partner Af-9 and is retained in all Hrx-Enl and Hrx-Af9 fusion proteins . Thus, the leukemogenic contribution and transcriptional activation potential of Enl colocalize to its highly conserved carboxy terminus, suggesting that Hrx-Enl chimeric proteins mediate alterations in the transcription program of t(11;19)-bearing cells. Genes Dev, 1994 Sep 15, 8(18), 2176 - 87 A temperature-sensitive MEK mutation demonstrates the conservation of the signaling pathways activated by receptor tyrosine kinases; Hsu JC et al.; MEK, a dual specificity threonine/tyrosine kinase, has been postulated to be a convergent point for signaling from receptor protein tyrosine kinases (RTKs) and G-protein-coupled receptors . In contrast to yeast and mammalian cells where several MEKs have been isolated, only one Drosophila MEK (D-Mek) has been characterized to date . Previous studies have shown that D-Mek acts in the Torso RTK signaling pathway . To demonstrate that D-Mek also operates downstream of other RTKs, we generated a temperature-sensitive allele of D-mek (D-mekts) by site-directed mutagenesis based on the amino acid change of a yeast cdc2ts mutation . Using D-mekts, we show that in addition to its role in Torso signaling, D-Mek operates in the Sevenless and in the Drosophila epidermal growth factor RTK pathways . Because loss-of-function mutations in D-mek and the upstream receptors give rise to similar phenotypes, it suggests that D-mek is the only MEK activated by Drosophila RTKs . In addition, we demonstrate that different RTK pathways respond differently to alteration in D-Mek activity. Biochem J, 1994 Sep 15, 302 ( Pt 3), 687 - 94 Copper-dependent metabolism of Cu,Zn-superoxide dismutase in human K562 cells . Lack of specific transcriptional activation and accumulation of a partially inactivated enzyme; Steinkuhler C et al.; The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells . Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA . A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern . The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of {3H}lysine into acid-precipitable material . Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein . However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins . Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells . Pulse-chase experiments using {3H}lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation . From these results we conclude that: (i) unlike in yeast {Carri, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett . 278, 263-266} Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in intact K562 cells; and (iii) the metabolic stability of Cu,Zn-superoxide dismutase results in its relative accumulation under conditions that lead to increased protein turnover. Genomics, 1994 Sep 15, 23(2), 496 - 9 Isolation of a human YAC contig encompassing a cluster of UGT2 genes and its regional localization to chromosome 4q13; Monaghan G et al.; Previously we mapped the gene encoding a human bile acid UDP-glucuronosyltransferase (UGT2B4) to chromosome 4 . Here we report the mapping of two additional human UGT2B genes to chromosome 4 utilizing the polymerase chain reaction (PCR) and a panel of human/rodent somatic cell hybrid cell lines . A yeast artificial chromosome contig containing the UGT2B4, UGT2B9, and UGT2B15 genes was isolated, and pulsed-field gel electrophoresis and PCR revealed that several members of the human UGT2B gene subfamily are clustered within a 195-kb region of the YAC contig . These data permitted a provisional ordering of the genes as UGT2B9-UGT2B4-UGT2B15 . Fluorescence in situ hybridization analysis, using the YAC DNA, permitted the regional localization of this gene cluster to chromosome 4q13. Genomics, 1994 Sep 15, 23(2), 492 - 5 Organization of the region encompassing the human serum amyloid A (SAA) gene family on chromosome 11p15.1; Sellar GC et al.; The four members of the human serum amyloid A protein (SAA) gene family are clustered on human chromosome 11p15.1 . Three genes are differentially expressed and encode small apolipoproteins of M(r) 12-19 kDa: SAA1 and SAA2 encode the acute phase SAAs (A-SAAs), and SAA4 encodes the constitutively expressed SAA (C-SAA) . A fourth locus, SAA3, is a pseudogene . The human SAA gene family encompasses approximately 150 kb contained on a 900-kb yeast artificial chromosome contig . SAA1 and SAA2 are 15-20 kb apart and are arranged in divergent transcriptional orientations . SAA4 is 9 kb downstream of SAA2 and in the same orientation . SAA3 is 110 kb downstream of SAA4, and its relative orientation could not be determined . All genes known to be in the same human and mouse syntenic linkage group as SAA were mapped within the contig . Interphase FISH was used to orientate the region relative to the centromere: cen-LDHC-LDHA-SAA1-SAA2-SAA4-SAA3-TP H-D11S18- KCNC1-MYOD1-pter. Genomics, 1994 Sep 15, 23(2), 420 - 4 Isolation and chromosomal mapping of genomic clones encoding the human fatty acid synthase gene; Jayakumar A et al.; We have isolated and sequenced 0.5- and 3.6-kb cDNA clones that cover the N-terminal and carboxy-terminal regions, respectively, of the human fatty acid synthase . To localize the fatty acid synthase gene and to define its genomic structure, we have also isolated overlapping genomic clones by screening two human YAC libraries with PCR primers derived from the fatty acid synthase cDNA sequences . The DNA inserts in these human fatty acid synthase YACs hybridized with human synthase-specific cDNA probes . Using biotin-labeled Alu-PCR products of the human synthase YACs as probes for fluorescence in situ hybridization, we mapped the fatty acid synthase gene to chromosome 17q25 . We also screened a chromosome 17-specific cosmid library with human synthase cDNA probes and isolated 12 cosmids, all of which had EcoRI fragments in common . DNA sequencing of an amplified PCR product from the fatty acid synthase cosmids confirmed that these genomic clones contained expressed fatty acid synthase sequences . Furthermore, the results of Southern analyses suggested that a single 40-kb cosmid clone encompasses the entire coding region of the fatty acid synthase gene . The synthase gene is located on chromosome 17 near the q25 band, which is close to the telomere and could serve as an important marker in analysis of this chromosome. Genomics, 1994 Sep 15, 23(2), 408 - 19 Three genes in the human MHC class III region near the junction with the class II: gene for receptor of advanced glycosylation end products, PBX2 homeobox gene and a notch homolog, human counterpart of mouse mammary tumor gene int-3; Sugaya K et al.; Cosmid walking of about 250 kb from MHC class III gene CYP21 to class II was conducted . The gene for receptor of advanced glycosylation end products of proteins (RAGE, a member of immunoglobulin superfamily molecules), the PBX2 homeobox gene designated HOX12, and the human counterpart of the mouse mammary tumor gene int-3 were found . The contiguous RAGE and HOX12 genes were completely sequenced, and the human int-3 counterpart was partially sequenced and assigned to a Notch homolog . This human Notch homolog, designated NOTCH3, showed both the intracellular portion present in the mouse int-3 sequence and the extracellular portion absent in the int-3 . It thus corresponds to the intact form of a Notch-type transmembrane protein . About 20 kb of dense Alu clustering was found just centromeric to the NOTCH3. Genomics, 1994 Sep 15, 23(2), 338 - 43 Genomic organization and expressed sequences of the mouse extended H-2K region; Lai F et al.; The mouse major histocompatibility complex (MHC) has long been of great interest to many biologists because of not only its critical role in the immune system, but also its association with at least three embryonic lethal genes . Here, we present an analysis of the mouse extended H-2K region using YAC technology . Six new expressed sequences were identified, demonstrating that the high gene density previously described continues . Restriction mapping of a YAC clone extending proximal of the MHC region defined a CpG-rich region located up to 320 kb away from H-2K . The absence of any CpG-rich region for a distance spanning approximately 200 kb near the YAC's proximal end suggests that the high gene density probably diminishes at a distance of 360 kb away from H-2K . The description of genomic organization of both H-2K and the extended H-2K region provides insight into the characteristics of this whole region with respect to gene diversity and density. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 8797 - 801 Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53; Scheffner M et al.; The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway . The E6 protein binds to a cellular protein of 100 kDa termed E6-AP . The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2) . This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level . In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily . We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53. FEBS Lett, 1994 Sep 12, 351(3), 340 - 4 Activation of gastrin gene transcription in islet cells by a RAP1-like cis-acting promoter element; Simon B et al.; Gastrin transcription in islet cells is activated by a cis-regulatory sequence containing a binding site for the yeast transcription factor RAP1 . The DNA-protein interactions between RAP1 protein and the gastrin DNA element determined by methylation interference assays are identical to those of RAP1 and yeast genes . Point mutations in the gastrin RAP1 binding site, which abolished RAP1 binding, decreased transcriptional activation by this sequence . Islet cells revealed a DNA binding protein with RAP1-like binding specificity . These findings support the conclusion that gastrin transcription is activated in mammalian cells by a RAP1-like transcription factor. Cell, 1994 Sep 9, 78(5), 773 - 85 The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B; Palombella VJ et al.; We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B . The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation . The C-terminal region of p105 is rapidly degraded, leaving the N-terminal p50 domain . p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants . These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha . Thus, the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides, but also in the regulated processing of precursors into active proteins. J Cell Biol, 1994 Sep, 126(6), 1341 - 51 Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events; Ishida R et al.; ICRF-193, a novel noncleavable, complex-stabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T . Miki, T . Narita, R . Yui, S . Sato, K . R . Utsumi, K . Tanabe, and T . Andoh . 1991 . Cancer Res . 51:4909-4916) . With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated . Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes . In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by ICRF-193 . Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases . In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of cdc2 kinase, spindle apparatus reorganization and disassembly and reassembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase . Cells then continued through further cell cycle rounds, becoming polyploid and losing viability . This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast . The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes . In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes. Dev Biol, 1994 Sep, 165(1), 185 - 92 An ascidian homolog of SEC61 is expressed predominantly in epidermal cells of the embryo; Ueki T et al.; In eukaryotic cells, SEC61 protein is essential for protein translocation across the endoplasmic reticulum membrane . Because of the ubiquitous nature of the protein, the expression of the corresponding gene(s) during embryogenesis has not received much attention . We found that an ascidian homolog of SEC61 is expressed predominantly in embryonic epidermal cells . A cDNA clone for Halocynthia roretzi SEC61 (HRSEC61) gene contains a single open reading frame that encodes a polypeptide of 475 amino acids . The degree of amino-acid identity was 87% between the ascidian and the dog and 55% between the ascidian and yeast . The HRSEC61 gene was transcribed maternally and zygotically . A low level of the corresponding mRNAs, about 2.3 kb in length, was found in eggs and early embryos . Zygotic mRNAs appeared after the gastrula stage, and the accumulation of the mRNAs was maximal at the neurula and tailbud stages . In situ hybridization of whole-mount specimens demonstrated that, although maternal mRNAs were distributed evenly in eggs and early embryos, the occurrence of zygotic mRNAs was predominant in differentiating epidermal cells . The predominant expression of HRSEC61 in embryonic epidermal cells was determined using cleavage-arrested embryos; those at the 16- or 32-cell stage expressed HRSEC61 in the presumptive epidermal cells of the animal hemisphere. J Neurosci, 1994 Sep, 14(9), 5338 - 51 Nuclear-encoded mitochondrial precursor protein: intramitochondrial delivery to dendrites and axon terminals of neurons and regulation by neuronal activity; Liu S et al.; Mitochondria contain hundreds of proteins, most of which are encoded by the nucleus . In neurons, distal dendrites and axon terminals can be separated from the nucleus by a great distance, and the mechanism by which precursor proteins reach distal neuronal processes is not well understood . While our previous study on cytochrome oxidase suggests a post-translational mechanism of delivery, it is not known whether precursor proteins reach their target processes before or after incorporation into mitochondria . In order to localize only precursor proteins and not the mature form of the subunit in neurons, we generated polyclonal antibodies against synthetic presequence polypeptides specific to nuclear-encoded subunit IV precursor protein of rat brain cytochrome oxidase . We found that the precursors were located not only in neuronal cell bodies, but also in dendrites and axon terminals . This indicates that the conversion of these precursors to their mature form is not confined to the cell body but occurs in dendrites and axons as well . At the electron microscopic level, an overwhelming majority of immunoreaction product was found within mitochondria, suggesting that precursor proteins are transported to neuronal processes mainly within mitochondria, and that their half-lives are much longer in neurons than in yeast and rat hepatocytes . The precursor pool was downregulated in the rat superior colliculus after monocular enucleation, indicating that precursor synthesis and/or degradation is regulated by neuronal functional activity . These results also suggest that local functional demands may play an important role in controlling the processing of precursors and the assembly of holoenzymes in dendrites and axon terminals . This allows neurons to regulate enzyme levels locally, precisely, and rapidly. J Am Acad Dermatol, 1994 Sep, 31(3 Pt 2), S25 - 30 Current therapy of dermatophytosis; Degreef HJ et al.; In the past dermatophytes were treated with topical agents or, in the case of more recalcitrant or extensive disease, with oral antifungals (griseofulvin or ketoconazole) . Topical therapies may be effective in many cases, but they have limitations . They may be viewed as inconvenient by the patient, thereby affecting compliance . Therapy with early oral antifungals entails long treatment periods until complete cure is obtained . For ketoconazole rare but serious side effects can occur, particularly with prolonged use . Griseofulvin is still the drug of choice for the treatment of tinea capitis of the Microsporum type . In recent years a few new antimycotic agents have been developed for systemic therapy of superficial fungal infections . Itraconazole is a broad-spectrum triazole . Fluconazole belongs to the same chemical class and was used mainly in systemic yeast infections and mucosal candidosis . Terbinafine is an allylamine and has been found to be effective and safe in brief therapy of dermatophyte infections . Short-duration therapy of most dermatophyte infections is also possible with itraconazole . The high and specific activity against the causative agents, together with their pharmacokinetic properties, explains the good results obtained with these new drugs and their improved safety profile . Their mode of action, pharmacokinetics, and treatment schedules will be discussed. J Am Acad Dermatol, 1994 Sep, 31(3 Pt 2), S18 - 20 Pityrosporum infections; Faergemann J; Pityrosporum ovale is a lipophilic yeast that is part of the normal human adult cutaneous flora . It is both a saprophyte and an opportunistic pathogen associated with pityriasis versicolor, Pityrosporum folliculitis, seborrheic dermatitis, and some forms of atopic dermatitis . Systemic infections have also been described . In this article the diagnosis and management of pityriasis versicolor, Pityrosporum folliculitis, seborrheic dermatitis, and atopic dermatitis will be discussed. Cell Immunol, 1994 Sep, 157(2), 393 - 402 Evidence for involvement of beta-glucan-binding cell surface lectins in human natural killer cell function; Duan X et al.; We have studied the effects of yeast cell wall derivatives (zymosan and particulate beta-glucan), on the cytolytic effector function of human natural killer cells . Both zymosan and particulate beta-glucan were found to inhibit the NK-cell-mediated killing of K562, Molt-4, U937, and HL60 tumor cells . Zymosan also inhibited the IL-2-dependent proliferation of NK cells, suggesting that some component of the yeast cell wall delivers a down-modulatory signal affecting multiple NK cell functions . NK cell surface molecules capable of binding both zymosan and Sepharose-immobilized pustulan (linear 1,6-beta-D-glucan, a carbohydrate component of zymosan and particulate beta-glucan) were identified in detergent lysates prepared from surface iodinated NK cells . Our results suggest that NK cells express cell surface beta-glucan-binding lectins that may contribute to NK-cell-mediated natural cytotoxicity. Mol Cell Biol, 1994 Sep, 14(9), 6287 - 96 Structure of REC2, a recombinational repair gene of Ustilago maydis, and its function in homologous recombination between plasmid and chromosomal sequences; Rubin BP et al.; Mutation in the REC2 gene of Ustilago maydis leads to defects in DNA repair, recombination, and meiosis . Analysis of the primary sequence of the Rec2 protein reveals a region with significant homology to bacterial RecA protein and to the yeast recombination proteins Dmc1, Rad51, and Rad57 . This homologous region in the U . maydis Rec2 protein was found to be functionally sensitive to mutation, lending support to the hypothesis that Rec2 has a functional RecA-like domain essential for activity in recombination and repair . Homologous recombination between plasmid and chromosomal DNA sequences is reduced substantially in the rec2 mutant following transformation . The frequency can be restored to a level approaching, but not exceeding, that observed in the wild-type strain if transformation is performed with cells containing multiple copies of REC2. Mol Cell Biol, 1994 Sep, 14(9), 5756 - 65 Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor; Casanova J et al.; The ligand-binding domains of thyroid hormone (L-triiodothyronine {T3}) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions . Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor . GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR . This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor . A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 {GAL4-T3R(408)-VP16} is activated by unliganded receptor as well as by T3 . In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3 . The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family . In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies . Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3 . These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex. J Exp Med, 1994 Sep 1, 180(3), 1087 - 96 Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1; Wright A et al.; Immunoglobulin G (IgG) molecules are glycosylated in CH2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions . The carbohydrate attached to the IgG constant region is a complex biantennary structure . Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis . To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps . We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1 . The Pro-5-produced Ab (IgG1-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complement-mediated cytolysis, and affinity for Fc gamma RI . Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, C1q binding, and C1 activation . IgG1-Lec 1 also showed reduced but significant affinity for Fc gamma R1 receptors . The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma- or Pro-5-produced counterpart, with more being cleared during the alpha-phase and with more rapid clearance during the beta-phase . Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan . Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide . Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure . Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function. Oncogene, 1994 Sep, 9(9), 2691 - 8 Ras-15A protein shares highly similar dominant-negative biological properties with Ras-17N and forms a stable, guanine-nucleotide resistant complex with CDC25 exchange factor; Chen SY et al.; We show that expression of Ras-15A, previously shown to be a dominant-negative mutant in yeast, is a potent inhibitor of endogenous Ras protein function in mammalian cells . Expression of Ras-15A did not inhibit the growth of cells containing an oncogenic ras gene nor did it interfere with the ability of transiently expressed oncogenic ras or raf genes to activate transcription from a Ras-responsive ets1/AP-1 promoter . In contrast, expression of Ras-15A completely blocked growth of normal cells and activation of the ets1/AP-1 promoter by transiently overexpressed SOS1 and normal Ras proteins . These results suggest that Ras-15A, like Ras-17N, blocks endogenous Ras function by interfering with upstream activation of Ras proteins rather than downstream effects . To test whether Ras-15A and Ras-17N interfere with Ras function by blocking GDP-GTP exchange proteins, we examined their physical interaction with the CDC25 exchange protein . All three proteins formed stable complexes with CDC25 in the absence of guanine-nucleotides, but only Ras-15A was not released from CDC25 by physiological concentrations of GDP or GTP . These results establish that Ras-15A blocks the activation of normal Ras proteins by sequestering GDP-GTP exchange factors into non-productive complexes . In contrast, it would appear that the similar biological properties of Ras-17N are mediated by a reversible, competitive sequestration of exchange factors. Mamm Genome, 1994 Sep, 5(9), 566 - 71 A refined restriction map of YAC clones spanning the entire human dystrophin gene; Nobile C et al.; The enormous size of the human dystrophin gene (2300 kb) has so far hindered the analysis of its organization and the characterization at the genomic level of the deletion and duplication mutations causing Duchenne or Becker muscular dystrophy . A detailed physical map of the gene locus would considerably simplify these studies . We constructed a refined, long-range restriction map of the entire human dystrophin gene, using 12 overlapping YAC clones as DNA sources . The sites for six rare cutting enzymes (SfiI, NruI, EagI, BssHII, SacII, and NotI) were mapped by partial digest analysis of YACs over a region of 2600 kb, within a level of resolution of about 10 kb . Such a map provides the first detailed representation of the physical structure of the dystrophin gene . It will be useful for mapping unlocalized exons and, eventually, for the characterization of deletions and duplications leading to disease. Trends Pharmacol Sci, 1994 Sep, 15(9), 343 - 9 Dissection of eukaryotic transmembrane signalling using Chlamydomonas; Quarmby LM et al.; Novel insights and surprises are often generated when investigators choose an organism that permits a new approach to a problem . For example, secretory and cell-cycle mutants in yeast have provided quantum leaps in elucidating these processes . Similarly, genetic systems are providing exciting new insights into signal transduction . The 'green yeast' Chlamydomonas has the potential to be a particularly rich organism for genetic analysis of signal transduction because, although unicellular, it has several interesting behaviours, which are discussed in this article by Lynne Quarmby and Criss Hartzell . Phototaxis results from the transduction of a light signal received by the eyespot to changes in flagellar beat . The mating reactions, which culminate in the fusion of gametes, are initiated in response to adhesion of flagellar proteins . Deflagellation, or flagellar shedding, is an acute response to a variety of stimuli . Molecular genetic analysis of behavioural mutants is providing new directions for understanding signal integration and segregation. Microbiology, 1994 Sep, 140 ( Pt 9), 2475 - 9 Hydrocortisone-enhanced growth of Aspergillus spp.: implications for pathogenesis; Ng TT et al.; Aspergillus fumigatus and Aspergillus flavus are the most common cause of invasive mould infections worldwide and carry a high mortality . Corticosteroid therapy and Cushing's disease are associated with an increase in invasive aspergillosis . Corticosteroids impair immune function in mammals and, specifically, the conidicidal activity of human macrophages, which was thought to be sufficient explanation for this increased risk . However, we have found a 30-40% increase in growth rate of A . fumigatus and A . flavus exposed to pharmacological doses of hydrocortisone (a human glucocorticoid), suggesting an alternative or additional mechanism for the association . No significant effect was observed with other human steroids such as testosterone, oestradiol or progesterone, though a smaller (21%) but significant growth rate increase was obtained with the fungal sterol ergosterol . The presence of a ligand/receptor system is therefore possible in pathogenic Aspergillus spp . Although corticosterone-binding proteins have been identified in some yeast species, a demonstrable physiological effect has been lacking . Interruption of the putative ligand/receptor interaction could have a major effect on the growth and pathogenicity of A . fumigatus, providing opportunities for the development of alternative therapeutic strategies to those currently available. Tijdschr Diergeneeskd, 1994 Sep 1, 119(17), 500 - 2 {Sporotrichosis in a horse}; Greydanus-van der Putten SW et al.; A 9-year old male Arabian horse was referred to the Department of Large Animal Surgery of the University of Utrecht because of multiple nodules on the inner side of the right hind leg . The nodules seemed to follow a cutaneolymphatic pattern . Histopathology of a nodule showed a granulomatous inflammation with the presence of multinucleated giant cells . In PAS- and Grocott-stained sections, spheroid yeast-like organisms with some budding were found throughout the tissue . A preliminary diagnosis of sporotrichosis was made . A fresh nodule was cultured and the presence of Sporothrix c.f . schenckii was confirmed . Antifungal therapy was started, which stopped the extension of the granulomata . Sporotrichosis is a rare disease in Europe, but it is quite common in the southern part of the U.S.A. Nucleic Acids Res, 1994 Sep, 22(17), 3566 - 8 Yale database for DNA sequence changes in mutagenesis; Hutchinson F et al.; The Yale database contains sequence changes in mutations induced in a number of bacterial, mammalian and yeast genes . It contains data in electronic form on more than 17,000 mutations (July, 1994), is periodically updated, and is available without cost on Internet and on diskettes . Researchers are invited to contribute additional results; a data entry program, MUSTIN, is provided to facilitate adding new data and to minimize errors. Plant J, 1994 Sep, 6(3), 447 - 55 Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones; Klein M et al.; As part of the worldwide efforts at molecular analysis of Arabidopsis thaliana as a model plant the complete structure of the mitochondrial genome has been determined . The mitochondrial DNA molecules were mapped by restriction fragment analysis of more than 300 cosmid clones and purified mitochondrial DNA . The entire genome of 372 kb is contained in three different configurations of circular molecules and is split into two additional subgenomic molecules of 234 kb and 138 kb, respectively . These arrangements result from recombinations of the two sets of repeats present in combinations of inverted and/or direct orientation . Alignment of YAC clones confirms the in vivo presence of continuous DNA molecules of more than 300 kb in A . thaliana mitochondria . The presence of this comparatively large mitochondrial genome in a plant with one of the smallest nuclear genomes shows that different size constraints act upon the different genomes in plant cells. Plant Cell, 1994 Sep, 6(9), 1289 - 99 An Arabidopsis peptide transporter is a member of a new class of membrane transport proteins; Steiner HY et al.; An Arabidopsis peptide transport gene was cloned from an Arabidopsis cDNA library by functionally complementing a yeast peptide transport mutant . The Arabidopsis plant peptide transporter (AtPTR2) allowed growth of yeast cells on dipeptides and tripeptides but not peptides four residues and higher . The plant peptide transporter also conferred sensitivity to a number of ethionine-containing, toxic peptides of chain length three or less and restored the ability to take up radiolabeled dileucine at levels similar to that of the wild type . Dileucine uptake was reduced by the addition of a variety of growth-promoting peptides . The sequence of a cDNA insert of 2.8 kb indicated an open reading frame encoding a 610-amino acid polypeptide (67.5 kD) . Hydropathy analysis predicted a highly hydrophobic protein with a number of potential transmembrane segments . At the amino acid level, the Arabidopsis plant peptide transporter shows 24.6, 28.5, and 45.2% identity to the Arabidopsis nitrate-inducible nitrate transporter (CHL1), the rabbit small intestine oligopeptide transporter (PepT1), and the yeast peptide transporter (Ptr2p), respectively, but little identity to other proteins known to be involved in peptide transport . Root growth of Arabidopsis seedlings exposed to ethionine-containing toxic peptides was inhibited, and growth was restored by the addition of certain peptides shown to compete with dileucine uptake in yeast expressing the Arabidopsis transport gene . Consistent with the observed inhibition of root growth by toxic peptides, the peptide transporter is expressed in the roots of Arabidopsis seedlings . This study represents the characterization of a plant peptide transporter that is a member of a new class of related membrane transport proteins. J Steroid Biochem Mol Biol, 1994 Sep, 50(5-6), 313 - 8 Equilibrium hormone binding to human estrogen receptors in highly diluted cell extracts is non-cooperative and has a Kd of approximately 10 pM; Salomonsson M et al.; It is generally accepted that the Kd for hormone binding to estrogen receptors in extracts ranges between 0.1-1 nM and that binding displays positive cooperativity due to formation of homodimers . After carefully optimizing assay procedures, to diminish ligand depletion phenomena and to fully control recoveries, we find a single class of non-interacting high affinity hormone binding sites with a Kd of approx . 10 pM . Ligand depletion was avoided by decreasing receptor concentrations to 5-8 pM . We were therefore obliged to employ radioiodinated estradiol as a probe as the specific radioactivity of tritiated estradiol was too low to maintain the accuracy of the binding assay . Human estrogen receptor extracted from the MCF7 cell line and recombinantly produced (in yeast) wild-type human receptor have identical equilibrium hormone binding characteristics. Mol Carcinog, 1994 Sep, 11(1), 13 - 8 Use of representational difference analysis for the identification of mdm2 oncogene amplification in diethylstilbestrol-induced murine uterine adenocarcinomas; Risinger JI et al.; Exposure in utero to the synthetic estrogen diethylstilbestrol (DES) is associated with the subsequent development of reproductive-tract malignancies in female offspring . To search for the genetic targets of DES, representational difference analysis was used to compare genomic DNA from DES-associated mouse uterine adenocarcinoma cells with genomic DNA from normal CD-1 mouse tissue . Several difference clones were obtained, all of which recognized rearranged and amplified sequences in tumor compared with normal DNA . One of these difference fragments mapped to a region of mouse chromosome 10 that includes the mdm2 oncogene . Amplification and overexpression of mdm2 was found in all three early-passage cell lines established from independent DES-associated cancers . These findings demonstrate the potential power of representational difference analysis in cancer research and suggest a genetic mechanism for DES-induced carcinogenesis. Clin Exp Immunol, 1994 Sep, 97(3), 361 - 6 Enhanced activation of human T cell clones specific for virus-like particles expressing the HIV V3 loop in the presence of HIV V3 loop-specific polyclonal antibodies; Peifang S et al.; Recombinant virus-like particles (VLP), formed by the yeast Ty p1 protein, carrying the HIV gp120 V3 loop on their surface (V3-VLP) have been tested in vitro for immunogenicity and antigenicity by using VLP p1-specific human CD4+ T cell lines and clones . VLP-specific human T cell lines and clones were generated from normal individuals, indicating that VLP-specific precursor cells present in the peripheral lymphocyte pool can be induced to expand clonally upon antigen challenge in vitro, in the absence of previous immunization . It was also shown that V3-specific polyclonal antibodies enhance V3-VLP-induced activation of VLP-specific T cell clones . Antibody-dependent potentiation has been shown previously in other antigen systems, and it depends on enhanced uptake of complexed antigen by Fc receptor-positive antigen-presenting cells . Since in this case antigen is internalized by presenting cells as a complex, it can be inferred that a similar event of antibody-mediated antigen uptake can take place with V3-specific B cells, resulting in presentation by the B cells of T helper epitopes derived from processing of the VLP p1 moiety . This suggests that T helper cells specific for the carrier VLP p1 protein can be activated to provide help to V3-specific B cells in the presence of the appropriate antigen construct. Cancer Biochem Biophys, 1994 Sep, 14(2), 123 - 31 Canine U2 snRNA gene: nucleotide sequence, characterization and implications in RNA processing and cancer biology; Verma M et al.; Abnormal RNA processing (splicing) may lead a cell to become cancerous . Transcription of a gene starts in the nucleus where genomic DNA is converted to precursor RNA by removing introns and joining exons . Splicing, mediated by small nuclear RNA (snRNA) and nuclear proteins, is tightly regulated during growth and development . U2 snRNAs are small, stable RNAs located in the nucleus of eukaryotic cells that recognize the branch point of the intron-exon junction . We describe here the organization of DNA sequences complementary to canine U2 snRNA . From a genomic library we isolated one recombinant containing the U2 gene . Southern analysis revealed that the canine species possesses only 3 to 5 U2 snRNA genes or very closely related sequences . The size of the U2 gene is 125 nt whereas in rat, Drosophila, trypanosome and yeast it is 189, 234, 141, and 192 nt respectively . The nucleotide sequence showed 82, 78, 72 and 95% homology with rat, Drosophila, yeast, and trypanosome U2 snRNA, respectively . The sensitivity of U2 snRNA towards alpha-amanitin suggests that it is transcribed by RNA polymerase II . The conserved nucleotide sequences which have been implicated in heterogeneous nuclear RNA splicing have been identified . The implications of the knowledge gained through above studies in cancer biology are discussed. Methods Find Exp Clin Pharmacol, 1994 Sep, 16(7), 513 - 8 Genetically engineered in vitro systems for biotransformation studies; Doehmer J et al.; In order to understand cytochrome P450-mediated metabolism of xenobiotics such as drugs and pollutants, several cell systems are genetically engineered for metabolic competence by cloning cDNAs encoding cytochrome P450 and other enzymes and by heterologous expression in bacterial, yeast, and mammalian cells . Genetically engineered cell systems are defined for the cDNA enzyme function . In conjunction with cell intrinsic properties, these genetically engineered cell systems can be used for the assessment of metabolism-dependent pharmacological and/or toxicological effects. Mol Endocrinol, 1994 Sep, 8(9), 1182 - 92 Sry is a transcriptional activator; Dubin RA et al.; The SRY gene functions as a genetic switch in gonadal ridge initiating testis determination . The mouse Sry and human SRY open reading frames (ORFs) share a conserved DNA-binding domain (the HMG-box) yet exhibit no additional homology outside this region . As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and mouse SRY ORFs contain a nuclear localization signal . The mouse Sry HMG-box domain selectively binds the sequence NACAAT in vitro when challenged with a random pool of oligonucleotides and binds AACAAT with the highest affinity . When put under the control of a heterologous promotor, the mouse Sry gene activated transcription of a reporter gene containing multiple copies of the AACAAT binding site . Activation was likewise observed for a GAL4-responsive reporter gene, when the mouse Sry gene was linked to the DNA-binding domain of GAL4 . Using this system, the activation function was mapped to a glutamine/histidine-rich domain . In addition, LexA-mouse Sry fusion genes activated a LexA-responsive reporter gene in yeast . In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation . These studies suggest that both the human and the mouse SRY ORFs encode nuclear, DNA-binding proteins and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains. Genomics, 1994 Sep 1, 23(1), 85 - 93 Cloning, sequencing, and mapping of the human chromosome 14 heat shock protein gene (HSPA2); Bonnycastle LL et al.; A genomic clone for the human heat shock protein (HSP) 70 gene located on chromosome 14 was isolated and sequenced . The gene, designated HSPA2, has a single open reading frame of 1917 bp that encodes a 639-amino acid protein with a predicted molecular weight of 70,030 Da . Analysis of the sequence indicates that HSPA2 is the human homologue of the murine Hsp70-2 gene with 91.7% identity in the nucleotide coding sequence and 98.2% in the corresponding amino acid sequence . HSPA2 has less amino acid homology to other members of the human HSP70 gene family, 83.3% to the heat-inducible HSP70-1 gene and 86.1% with the human heat shock cognate gene HSC70 . HSPA2 is constitutively expressed in most tissues, with very high levels in testis and skeletal muscle . Significant but lower levels are also expressed in ovary, small intestine, colon, brain, placenta, and kidney . A yeast artificial chromosome (YAC) clone containing HSPA2 (YAC741H4) that also contained the polymorphic marker D14S63 was identified . This 670-kb YAC was mapped to 14q24.1 by fluorescence in situ hybridization (FISH) . Subsequent two-color FISH and genetic mapping placed HSPA2/D14S63 proximal to the markers D14S57 and D14S77. Genomics, 1994 Sep 1, 23(1), 75 - 84 Isolation and mapping of human chromosome 21 cDNA: progress in constructing a chromosome 21 expression map; Cheng JF et al.; We have isolated 175 cDNA clones from a fetal brain library by direct cDNA selection using genomic DNA isolated from pools of human chromosome 21 (HC21) cosmids . DNA sequences have revealed that 16 of these cDNA clones contain overlapping sequences . Of the other 159 cDNA sequences, 10 match previously identified HC21 genes, and 9 match previously determined cDNA sequences, including the Wilms tumor related transcript (QM), the human testican cDNA, the mammalian calponin cDNA, and 6 anonymous expressed sequence tags . All isolated cDNAs were hybridized to their corresponding cosmids, which suggests that they originated from HC21 . We have localized 92 cDNA clones to previously reported HC21q YACs . The remaining unmapped cDNAs contain either sequences not included in the isolated HC21q YACs or sequences that hybridize to yeast DNA . The cDNAs not included in the YACs should be useful in isolating new YACs to bridge the gaps . PCR primers were derived from 4 novel cDNA sequences that had been mapped to the YACs in the suspected Down syndrome region and used in RT-PCR analysis . All 4 primer sequences amplified RNA fragments with the expected sizes, suggesting that these sequences could be used for expression analysis . The construction of a chromosome 21 cDNA map not only is important in the refinement of physical maps, but also will identify a set of genes in the disease regions for detailed characterization. Genomics, 1994 Sep 1, 23(1), 30 - 5 Genomic organization of the human VP16 accessory protein, a housekeeping gene (HCFC1) mapping to Xq28; Frattini A et al.; The region between DXS52 and Factor VIII gene in the human Xq28 chromosomal band contains a G+C-rich isochore to which many genes have been mapped . We report here the isolation and characterization of a transcript mapping about 50 kb telomeric from the vasopressin type 2 receptor gene in a 180-kb YACs/cosmid contig containing the L1CAM gene at its centromeric end . The determined transcribed sequence from a human fetal brain library is identical to that of the recently identified accessory protein HCFC1 (host cell factor, also called C1) that activates herpes simplex virus VP16 (alpha TIF) transactivator protein for association with the octamer motif-binding protein Oct-1 (Cell 74: 115, 1993) . The gene is expressed in a ubiquitous pattern and a larger transcript of approximately 10 kb is present in all the tissues tested, while an alternatively spliced RNA of approximately 8.0 kb is present in muscle and heart tissues . Genomic sequencing allowed us to determine that the sequenced transcript is assembled from 26 exons spread over a relatively small genomic region of approximately 24 kb . This alllowed us to determine that a previously reported cDNA clone arises from the splicing out of an internal portion of exon 8 which does not change the reading frame . All together these results raise the possibility that alternative mRNA processing could partly contribute to the diversity of the polypeptide HCFC1 family in a subset of tissues. Genomics, 1994 Sep 1, 23(1), 23 - 9 A YAC contig spanning the nevoid basal cell carcinoma syndrome, Fanconi anaemia group C, and xeroderma pigmentosum group A loci on chromosome 9q; Morris DJ et al.; Nevoid basal cell carcinoma syndrome (NBCCS, Gorlin syndrome) is an autosomal dominant disorder, characterized primarily by multiple basal cell carcinomas, epithelium-lined jaw cysts, and palmar and plantar pits, as well as various other features . Loss of heterozygosity studies and linkage analysis have mapped the NBCCS gene to chromosome 9q and suggested that it is a tumor suppressor . The apparent sensitivity of NBCCS patients to UV and X-irradiation raises the possibility of hypersensitivity to DNA-damaging reagents or defective DNA repair being etiological in the disorder . The recent mapping of the Fanconi anaemia group C (FACC) and xeroderma pigmentosum complementing group A (XPAC) genes to the same region on 9q has led us to begin the molecular dissection of the 9q22-q31 region . PCR analysis of the presence or absence of 10 microsatellite markers and exons 3 and 4 of the XPAC and FACC genes, respectively, allowed us to order 12 YACs into an overlapping contig and to order the markers as follows: D9S151/D9S12P1-D9S12P2-D9S197-D9S196-D9 S280-FACC-D9S287/XPAC-D9S180-D9S6-D9 S176 . Sizing of the YACs has provided an initial estimate of the size of the NBCCS candidate region between D9S12 and D9S180 to be less than 1.65 Mb. Genomics, 1994 Sep 1, 23(1), 178 - 84 Genetic map of the fused locus on mouse chromosome 17; Rossi JM et al.; Fused (Fu) is a dominant mutation in mice resulting in the asymmetry and fusion of tail vertebrae in heterozygotes . Fu/Fu homozygotes are often viable and can exhibit a duplication of the terminal tail vertebrae resulting in bifurcated tails . There are two more severe alleles at Fu, Kinky (FuKi) and Knobbly (FuKb), which die between 9 and 10 days of gestation as homozygotes, exhibiting a duplication of the embryonic axis, leading to incomplete or complete twinning . To define the precise map position of the FuKi mutation on mouse Chromosome 17, a 983-animal (FuKi tf x Mus spretus)F1 x +tf/+tf interspecific backcross was generated and scored for FuKi, another tightly linked visible marker tufted (tf), and five linked molecular loci, D17MIT18, D17Leh54, D17Aus57, Hba-ps4, and Pim1 . The order and genetic distances between the markers were determined to be centromere-D17MIT18-5.79 cM-D17Leh54-0.85 cM-D17Pri6-0.12 cM-D17Pri7-0.12 cM-Hba-ps4-1.20 cM-D17Pri8-0.48 cM-tf-2.05 cM-Pim1 . The FuKi gene could not be genetically separated from three molecular markers, D17Pri6, D17Pri7, and Hba-ps4 . Yeast artificial chromosome clones that contain these tightly linked markers have been isolated to form a contig that contains FuKi . Recombination breakpoints generated through the interspecies backcross were mapped onto the contig and demonstrate that recombination in this region is not random. Genomics, 1994 Sep 1, 23(1), 151 - 7 Mapping of human immunoglobulin heavy chain variable gene segments outside the major IGH locus; Wintle RF et al.; Physical mapping of the human immunoglobulin heavy chain gene cluster (IGH) on chromosome 14 has previously shown that the locus includes at least 63 variable region (VH) gene segments . Fifteen VH gene segments are located on six NotI DNA restriction fragments that are not within the mapped region of IGH . We have used human/rodent somatic cell hybrid lines to map these gene segments, as it was previously not proven that they are located in the chromosome 14 IGH locus . Four gene segments map to human chromosome 16 and two to chromosome 15 . Apparently, four of the six NotI fragments, representing 11 VH gene segments, are not located within the chromosome 14 IGH locus . In addition, we have demonstrated that a YAC containing a functional human telomere, and mapping to 14qter, is located at the telomeric end of the IGH gene cluster physical map and contains at least four VH gene segments . This YAC is collinear with the existing physical map of genomic DNA . We conclude that our original physical map of IGH represents almost the entire locus on chromosome 14 and that the 11 gene segments newly mapped are not part of the functional IGH locus. Genomics, 1994 Sep 1, 23(1), 132 - 7 Physical map of mouse chromosome 17 in the region relevant for positional cloning of the Hybrid sterility 1 gene; Trachtulec Z et al.; Hybrid sterility 1 (Hst1) is the major gene responsible for sterility of male hybrids between Mus musculus and certain laboratory strains . Thus, Hst1 is of importance in studying both postreproductive isolation of closely related species and male fertility . It has been mapped to mouse chromosome 17 in the region corresponding to the third inversion of the t haplotypes . The aim of the present study was to construct a physical map of the Hst1 region as the first step in an effort to clone the gene . Three yeast artificial chromosome (YAC) libraries (Princeton, Whitehead, and ICRF) were screened with polymerase chain reaction (PCR) oligonucleotide primers and DNA probes specific for loci previously mapped into the region of the third inversion . The isolated YAC clones were restriction mapped and arranged into contigs . Sixteen YAC clones were arranged into a single contig encompassing a region approximately 2000 kb long based on restriction mapping of highly overlapping but independently derived YAC clones . Five new loci in the region of the third inversion were mapped and the order and approximate physical distances of 12 loci established in this contig . The Hst1 gene maps approximately 0.2 cM proximal to the D17Ph1 locus encompassed by the YAC contig . Since the contig extends at least 1200 kb proximal to D17Ph1, it should contain the Hst1 gene. Comput Appl Biosci, 1994 Sep, 10(5), 465 - 70 Identification of a set of frequent decanucleotides in plants and in animals; Scapoli C et al.; We studied the frequency distribution of 1,048,576 oligonucleotides 10 bp long in a sample of 1.961 Mbase of genes from plants, made of 635 sequences extracted from GenBank 71.0, with the aim of detecting transcription control signals . Among all decamers, 3255, or 0.3%, had a frequency 10 times higher than the mean and were subjected to further statistical analysis . For each of the 3255 decamers (parents), we counted the individual frequencies of the 30 decamers (progeny) differing from the parent by one base mutation, and calculated two variance/mean chi-squares for the progeny, with and without the parent decamer . By studying the distribution of the ratio between the two chi-squares we observed that out of 3255 decamers > 10 times frequent than average, 432 had a chi-square ratio > 1.9 . In this residual set, which corresponds to < 0.04 per cent of all possible decamers, only 15 known eukaryotic transcription control elements were found; on the other hand, it included 29 decanucleotides that matched with decanucleotides of a set of Drosophila, 24 with a set from mammals, 13 with a set from yeast and four with a set of viruses--all sets identified with the statistical procedures here described . These decanucloetides are highly repetitive and seem to be present throughout all higher organisms, whereas they are uncommon in mammalian viruses. Chromosoma, 1994 Sep, 103(5), 324 - 30 Interstitial deletions of repetitive DNA blocks in dicentric human Y chromosomes; Kohler MR et al.; Cytogenetic analysis of aberrant human Y chromosomes was done by fluorescence in situ hybridization (FISH) with Y specific repetitive DNA probes . It revealed an interstitial deletion of different DNA blocks in two dicentric chromosome structures . One deletion includes the total alphoid DNA structure of one centromeric region . The second deletion includes the total repetitive DYZ5 DNA structure in the pericentromeric region of one short Y arm . Both dicentric Y chromosomes were iso(Yp) chromosomes with break and fusion point located in Yq11, the euchromatic part of the long Y arm . Their phenotypic appearance was "abnormal", resembling small monocentric Yq-chromosomes in metaphase plates . Mosaic cell lines, usually included in karyotypes with dicentric Y chromosomes, were not observed . It is assumed that both deletion events suppress the kinetochore activity in one Y centromeric region and thus stabilize its dicentric structure . Local interstitial deletion events had not been described in dicentric human Y chromosomes, but are common in dicentric yeast chromosomes . This raises the question of whether deletion events in dicentric human chromosomes are rare or restricted to the Y chromosome or also represent a general possibility for stabilization of a dicentric chromosome structure in human. Curr Biol, 1994 Sep 1, 4(9), 828 - 30 Cell cycle . In and out of the cell cycle; Forsburg SL; Studies of fission yeast are shedding light on how the same genes allow cells to respond to their environment either by growing and proliferating mitotically or by arresting growth to allow differentiation and meiosis. Mycopathologia, 1994 Sep, 127(3), 151 - 7 A natural focus of Histoplasma capsulatum var . duboisii is a bat cave; Gugnani HC et al.; The natural reservoir of Histoplasma capsulatum var . duboisii, the etiological agent of histoplasmosis duboisii (African histoplasmosis) is not yet known . We report the isolation of H . capsulatum var . duboisii from soil admixed with bat guano and from the intestinal contents of a bat in a sandstone cave in a rural area, Ogbunike in Anambra State of Nigeria . Eight of 45 samples of soil admixed with bat guano yielded H . capsulatum var . duboisii . Of the 35 bats belonging to the species Nycteris hispida and Tadirida pumila examined, only one (N . hispida) yielded this fungus from its intestinal contents . Identification of the isolates as Histoplasma was confirmed by exoantigen tests and by mating with tester strains of H . capsulatum . In vitro conversion to large yeast from suggestive of H . capsulatum var . duboisii was obtained on brain heart infusion agar supplemented with sheep blood and glutamine or cysteine . Pathogenicity tests with mice for all the isolates confirmed their identity by the demonstration of large yeast forms (8-15 microns in diameter) within giant cells in the infected tissues . Investigations on the possible occurrence of human infections in the area are in progress. Mycopathologia, 1994 Sep, 127(3), 145 - 9 Airborne fungal load in agricultural environment during threshing operations; Uddin N et al.; The effect of threshing operations on the fungal spora of farm air, has been studied with the culture plate method . Air samples were taken before, during and after the threshing of paddy and wheat grains, at the end of the respective crop seasons . An immediate local increase in the airborne fungal load was observed during threshing operations . In case of wheat the high rise was due to Alternaria tenuissima, followed by Drechslera sp . and Cladosporium herbarum . In 'Boro' variety of paddy, the high count was contributed by Alternaria humicola and A . tenuissima, while in 'Aman' variety, Helminthosporium oryzae was the dominant type followed by C . herbarum and unidentified yeasts . Aspergillus spp . were observed in significant numbers before the operation started; their occurrence was negligible during threshing . A considerable number of yeast was observed, particularly during threshing and after threshing operations. Mycoses, 1994 Sep-Oct, 37(9-10), 313 - 6 Comparative evaluation of the Premier enzyme immunoassay, micro-immunodiffusion and complement fixation tests for the detection of Histoplasma capsulatum var . capsulatum antibodies; Sekhon AS et al.; A total of 178 sera, including 68 from proven cases of histoplasmosis (65 positive for the presence of Histoplasma capsulatum var . capsulatum antibodies and three positive for antigen), 93 from patients with suspected histoplasmosis but with no laboratory evidence of H . capsulatum var . capsulatum infection, 14 from humans with heterologous fungal and non-fungal infections and three from normal individuals, were tested for IgG H . capsulatum antibodies and M or M and H precipitins by enzyme immunoassay (EIA) (Meridian Diagnostics, Cincinnati, OH, USA) and microimmunodiffusion (MID) respectively . Sixty-three of the 68 histoplasmosis case sera demonstrated IgG antibody, and 65 of 68 demonstrated the presence of specific precipitins in the MID test . Nine positive case sera, when tested with the Laboratory Branch complement fixation (LBCF) test, reacted positively to whole yeast and histoplasmin antigens (titres 1:8 to 1:512) . Three histoplasmosis case sera repeatedly tested negative for IgG, specific precipitins and complement-fixing antibodies, whereas they were positive for Histoplasma antigen . Eighteen of 95 sera from patients without evidence of histoplasmosis demonstrated IgG antibody in the EIA only . Among these positive sera, three out of three cases of aspergillosis and three out of five cases of blastomycosis were confirmed . Sera from HIV-infected and healthy individuals did not show IgG or M and/or H antibodies to H . capsulatum . Ninety-three sera were negative by both EIA and MID . The EIA for IgG was less sensitive (97%) than MID (100%) . The specificity of EIA and MID was 84% and 100% respectively. Mycoses, 1994 Sep-Oct, 37(9-10), 307 - 12 Polymerase chain reaction-mediated genotyping of Hortaea werneckii, causative agent of tinea nigra; Uijthof JM et al.; The black yeast Hortaea werneckii is known to be a causative agent of human tinea nigra but is also found in the environment . Strains from dissimilar sources were studied by polymerase chain reaction fingerprinting of nuclear DNA, using primers annealing to repetitive and random sequences . The seven groups found correspond to those known from restriction fragment length polymorphism (RFLP) studies of the mitochondrial DNA of the same strains . Two main groups contained strains from human as well as from non-human sources . The human strains did not cluster, but were randomly distributed over several populations . It was concluded that these strains are not pathogenic . The factor common to both niches is a relatively high salt concentration. Dev Comp Immunol, 1994 Sep-Oct, 18(5), 397 - 408 Specificity of a beta-glucan receptor on macrophages from Atlantic salmon (Salmo salar L.); Engstad RE et al.; This study was undertaken to study the specificity of a beta-glucan receptor on Atlantic salmon macrophages . Previous in vitro studies have shown that Atlantic salmon macrophages express a receptor that rapidly recognizes and mediates uptake of nonopsonized beta-glucan particles . The ingestion of particles was shown to be inhibited by preincubating the macrophages with glucans containing beta-1,3-linkages, but not by glucans containing other linkages . In the present study we have shown that small oligomers from formolyzed beta-glucan particles, and linear beta-1,3-linked oligomers with a degree of polymerization (DP) > or = 3, were efficient inhibitors of uptake of glucan particles . Oligomers from beta-1,6-linked pustulan, or small size oligomers with linkages other than beta-1,3, were not able to inhibit uptake of glucan particles . The inhibitory effect of laminarin and laminariheptaose was abolished by degrading the nonreducing terminal ends by sodium periodate treatment . The inhibitory effect of laminarin was regained by a complete Smith degradation; that is, periodate oxidation followed by reduction and hydrolysis . Modification of the reducing end of laminariheptaose had no effect on its ability to inhibit uptake . Furthermore, it was shown that periodate-oxidized glucan particles were not taken up by salmon macrophages, and that the uptake was regained when the particles were hydrolyzed to recover the nonreducing terminal end . Lastly, it was shown that endo-beta-1,6-glucanase treatment of the yeast glucan particles did not reduce uptake, confirming that beta-1,6-linkages are not involved in the recognition . These results suggest that Atlantic salmon macrophages possess a receptor that may recognize even very short beta-1,3-linked glucosyl chains extending from yeast cell walls. Genetika, 1994 Sep, 30(9), 1146 - 54 {Nucleotide sequence of DNA isolated from protein cores of rosette-like structures (elementary chromomeres) of mouse interphase chromosomes}; Glazkov MV et al.; Data are presented on the primary nucleotide sequence of 15 DNA fragments cloned from protein cores of rosette-like structures of mouse interphase chromosomes . According to data of a computer analysis, these DNA fragments represent protein-noncoding regions of interphase chromosomes, contain nucleotide sequences causing DNA strand bending, and contain a large amount of cis-regulatory transcriptional elements and sites closely related to sites of DNA topoisomerase II cleavage . Five clones demonstrated partial length homology with satellite DNA, short and long interspersed sequences, and extrachromosomal DNA from mouse thymocytes . Six clones showed homology with sites of initiation of replication of viruses, yeast, and mice . Six clones were found to contain consensus sequences for non-homologous recombination in somatic cells and six clones contained consensus sequences of human VTR1.1 loci. FEBS Lett, 1994 Aug 29, 351(1), 100 - 4 The role of lysine 99 of Thiobacillus versutus cytochrome c-550 in the alkaline transition; Ubbink M et al.; The methionine ligand of the heme iron in ferricytochrome c-550 from Thiobacillus versutus is replaced by another residue at high pH . This transition is similar to the alkaline transition in mitochondrial cytochrome c . To investigate the possible role of lysine 99 in this process, this residue has been mutated to a glutamate . The mutation causes the apparent pKa of the transition to decrease from 11.2 in wild type to 10.8 in Lys99Glu cytochrome c-550 . This destabilization of the native form is ascribed to the absence of the hydrogen bond between the epsilon-amine group of Lys99 and the carbonyl of Lys54 in the mutant protein . The 1H-NMR spectrum of Lys99Glu ferricytochrome c-550 at alkaline pH still shows resonance positions of the heme methyl peaks that are characteristic of the alkaline form . These results strongly suggest that Lys99 does not act as a ligand in the high-pH form, contrary to the case of yeast iso-1-cytochrome c . Evidence has been presented that in the latter protein the homologous Lys79 can act as a ligand in the alkaline form {1993, J . Am . Chem . Soc . 115, 7507-7508} . In the EPR spectrum of Lys99Glu cytochrome c-550 the species with Met-His coordination (gz = 3.27) is replaced by two forms with gz = 3.45 and 3.20 in the alkaline form (pH > or = 10.6) . At pH > 11 yet another form is observed with g-values 2.87, 2.18 and 1.60, tentatively identified as a species with a lysine-histidinate coordination of the heme iron. Cell, 1994 Aug 26, 78(4), 681 - 92 A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor; Rothe M et al.; Mutational analysis identified a C-terminal region of 78 amino acids within the cytoplasmic domain of the human 75 kDa tumor necrosis factor receptor (TNF-R2) that is required for signal transduction . This region was subsequently shown to mediate the interaction of cytoplasmic factors with TNF-R2 . Two of these factors were isolated and molecularly cloned using biochemical purification and the yeast two-hybrid system . TNF receptor-associated factor 1 (TRAF1) and TRAF2 are the first two members of a novel protein family containing a novel C-terminal homology region, the TRAF domain . In addition, TRAF2 contains an N-terminal RING finger motif . TRAF1 and TRAF2 can form homo- and heterotypic dimers . Our analysis indicates that TRAF1 and TRAF2 are associated with the cytoplasmic domain of TNF-R2 in a heterodimeric complex in which TRAF2 contacts the receptor directly . TRAF1 interacts with TNF-R2 indirectly through heterodimer formation with TRAF2. J Biol Chem, 1994 Aug 26, 269(34), 21762 - 9 The estrogen-inducible transferrin receptor-like membrane glycoprotein is related to stress-regulated proteins; Poola I et al.; It was previously shown that estrogen induces a membrane glycoprotein (molecular mass, 95 kDa) in the chicken oviducts, which exhibits several properties similar to transferrin receptors (Poola, I., and Lucas, J . J . (1988) J . Biol . Chem . 263, 19137-19146) . In the present study, we have further investigated its molecular and transferrin binding properties . We have sequenced several internal peptides isolated from the purified protein by endopeptidase Lys-C . We have found that it has a high degree of sequence homologies with those of chicken heat-shock protein (cHsp108), mouse endoplasmic reticulum protein (mERp99), hamster glucose-regulated protein (hagrp94), and human tumor rejection antigen (hTRAgp96), all of which are shown to be highly homologous to each other and to yeast hsp90 . We demonstrate here that the {35S}methionine-labeled immunoaffinity-purified estrogen-inducible membrane glycoprotein binds to the transferrin affinity columns similar to iron-modulated transferrin receptors . Indirect immunofluorescence microscopic studies indicate that it is an intracellular glycoprotein unlike transferrin receptors . We have isolated two molecular forms of the protein, with molecular masses of 116 and 104 kDa, by immunoaffinity column purification, immunoprecipitation, Western blotting, and pulse-chase labeling analyses . Both 116-and 104-kDa species bind transferrin . This protein can be induced by heat-shocking the oviduct cells at 45 degrees C for 3h and recovering at 37 degrees C for 2-3 h . It is also expressed in the human breast cancer cell lines, MCF-7 and T-47D . All these properties taken together strongly suggest that the estrogen-inducible membrane glycoprotein is a novel transferrin-binding protein, structurally related to the stress-regulated proteins. Nucleic Acids Res, 1994 Aug 25, 22(16), 3406 - 11 Characterization of four human YAC libraries for clone size, chimerism and X chromosome sequence representation; Nagaraja R et al.; Four collections of human X-specific YACs, derived from human cells containing supernumerary X chromosomes or from somatic cell hybrids containing only X human DNA were characterized . In each collection, 80-85% of YAC strains contained a single X YAC . Five thousand YACs from the various libraries were sized, and cocloning was assessed in subsets by the fraction of YAC insert-ends with non-X sequences . Cocloning was substantial, ranging up to 50% for different collections; and in agreement with previous indications, in all libraries the larger the YACs, the higher the level of cocloning . In libraries made from human-hamster hybrid cells, expected numbers of clones were recovered by STS-based screening; but unexpectedly, the two collections from cells with 4 or 5 X chromosomes yielded numbers of YACs corresponding to an apparent content of only about two X equivalents . Thus it is possible that the DNA of inactive X chromosomes is poorly cloned into YACs, speculatively perhaps because of its specialized chromatin structure. J Mol Biol, 1994 Aug 19, 241(3), 498 - 503 Genes encoding actin-related proteins of Drosophila melanogaster; Fyrberg C et al.; Recently, several laboratories have described proteins of yeasts, mammals and Drosophila melanogaster that are 35 to 55% identical to conventional actins, but, as yet, little is known about their functions . We have initiated a systematic study by using degenerate oligonucleotides specifying two highly conserved nucleotide-binding peptides of actin, in conjunction with polymerase chain reaction techniques, to isolate Drosophila genes that encode actin-related proteins . Here we summarize the isolation of four such genes and compare the sequences of the proteins that they encode . Computer searches of databases revealed that three of the encoded proteins are homologs of yeast or mammalian actin-related proteins, implying that the corresponding proteins participate in functions common to many cell types . The fourth gene encodes a novel protein that, apparently is expressed within testes . The four genes are located within the 14D, 53D, 66B and 87C subdivisions of polytene chromosomes. J Biol Chem, 1994 Aug 19, 269(33), 20857 - 65 Purification and characterization of human transcription factor IIIA; Moorefield B et al.; The 5S gene-specific transcription factor, TFIIIA, was purified approximately 35,000-fold from HeLa cell extracts using a combination of conventional and affinity chromatographic methods . A single polypeptide of apparent molecular mass 42-kDa cofractionates with both 5S DNA binding and 5S transcription activities, and was conclusively identified as human TFIIIA by its ability to direct specific 5S gene transcription in vitro following elution and renaturation from SDS-polyacrylamide gels . The DNase I protection pattern of the purified human factor on a human 5S gene is similar to the pattern previously observed with Xenopus TFIIIA . A Xenopus 5S gene, but not a yeast 5S gene, is able to effectively compete for binding of the human factor . In addition, we report a previously undetected immunological cross-reactivity between human and Xenopus TFIIIA . hTFIIIA is recognized specifically both by polyclonal antisera raised against Xenopus laevis TFIIIA as well as by a monoclonal antibody generated against the amphibian protein . These observations indicate that human TFIIIA is structurally related to the Xenopus oocyte factor and that the previous inability to detect human TFIIIA by immunological methods is due primarily to the low abundance of this factor in HeLa cell extracts. Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8268 - 72 The relationship between genetic and physical distances in the cloned a1-sh2 interval of the Zea mays L . genome; Civardi L et al.; A 470-kb segment from the long arm of chromosome 3 of Zea mays (inbred LH82), encompassing the a1-sh2 interval, was cloned as a yeast artificial chromosome . Comparison of the sizes of the restriction fragments generated from the cloned DNA fragment and from the DNA isolated from the maize inbred line LH82 established the colinearity of the a1-sh2 interval in these DNAs . By utilizing a chromosome fragmentation technique, a yeast artificial chromosome encompassing the a1-sh2 interval was separately fragmented at the a1 and sh2 loci . Comparison of the sizes of these fragmentation products established the physical distance between the a1 and sh2 loci to be 140 kb . Furthermore, these fragmentation experiments established the physical orientation of the a1 and sh2 genes relative to the maize centromere . The molecular cloning of the contiguous region between the a1 and sh2 loci made it possible to define the relationship between physical and genetic distances over a relatively large segment of the maize genome . In this interval, the relationship between physical and genetic distances is 1560 kb/centimorgan, which compares with 1460 kb/centimorgan for the entire maize genome, and 217 kb/centimorgan for a 1-kb segment within the a1 locus . Therefore, these findings are consistent with the hypothesis that genes per se are preferred sites for meiotic recombination rather than the hypothesis that genes reside in large recombinationally active segments of the genome. Biochem J, 1994 Aug 15, 302 ( Pt 1), 237 - 44 Structure and expression of the Drosophila ubiquitin-80-amino-acid fusion-protein gene; Barrio R et al.; In the fruitfly Drosophila, as in all eukaryotes examined so far, some ubiquitin-coding sequences appear fused to unrelated open reading frames . Two of these fusion genes have been previously described (the homologues of UBI1-UBI2 and UBI4 in yeast), and we report here the organization and expression of a third one, the DUb80 gene (the homologue of UBI3 in yeast) . This gene encodes a ubiquitin monomer fused to an 80-amino-acid extension which is homologous with the ribosomal protein encoded by the UB13 gene . The 5' regulatory region of DUb80 shares common features with another ubiquitin fusion gene, DUb52, and with the ribosomal protein genes of Drosophila, Xenopus and mouse . We also find helix-loop-helix protein-binding sequences (E-boxes) . The DUb80 gene is transcribed to a 0.9 kb mRNA which is particularly abundant under conditions of high protein synthesis, such as in ovaries and exponentially growing cells. FEBS Lett, 1994 Aug 15, 350(1), 55 - 60 Multiple nuclear localization signals of the B-myb gene product; Takemoto Y et al.; Nuclear entry of the B-myb gene product (B-Myb) is dependent on multiple nuclear localization signals (NLS's) . Mutagenesis of the putative NLS's of B-Myb has identified two separate NLS's, NLS1 and NLS2 . Each of the two NLS's is essential for efficient nuclear targeting . NLS2 contains two interdependent basic domains separated by 8 intervening spacer amino acids, and both basic domains are required for nuclear entry . Thus, NLS2 belongs to a class of bipartite NLS's . Like the NLS's in yeast transcription factor SW15, NLS2 contains a putative cdc2 kinase site . However, unlike the case of SW15, phosphorylation at this site did not affect the nuclear targeting of B-Myb. Blood, 1994 Aug 15, 84(4), 1309 - 13 Crystallization of recombinant hemoglobins with basic amino acid substitutions (Lys and Arg) at the beta 6 position; Adachi K et al.; We have produced recombinant hemoglobins (rHbs) alpha 2 beta 2(6Glu-->Lys) (rHb beta E6K) and alpha 2 beta 2(6Glu-->Arg) (rHb beta E6R) using a yeast expression system coupled with a polymerase chain reaction (PCR)-based mutagenesis strategy for studies focused on defining determinants that facilitate crystallization of Hb C (alpha 2 beta 2(6Lys)) . rHb beta E6K had the same electrophoretic mobility as native human Hb C, whereas rHb beta E6R migrated slightly slower than Hb C on cellulose acetate electrophoresis . The carbonmonoxy (CO) forms of rHb beta E6K and rHb beta E6R formed tetrahedral crystals in vitro in 2.3 mol/L phosphate buffer just like native Hb C . The Hb concentration required for crystallization of CO-rHb beta E6R was lower than that of CO-rHb beta E6K, suggesting that stronger basic amino acids at the beta 6 position accelerate crystallization of Hb . However, the size of rHb beta E6R crystals was smaller than that of rHb beta E6K . Crystallization of native Hb C and both rHbs was inhibited by Hb F . These results suggest that alpha 2 beta gamma-heterohybrids that have basic amino acids at the beta 6 position behave similarly and are unable to crystallize like Hb C. J Immunol, 1994 Aug 15, 153(4), 1769 - 77 Reconstitution of antibody-dependent phagocytosis in fibroblasts expressing Fc gamma receptor IIIB and the complement receptor type 3; Krauss JC et al.; In this study, we test the hypothesis that co-expression of both the complement receptor type 3 (CR3; CD11b/CD18) and Fc gamma receptor type IIIB (Fc gamma RIIIB) (CD16) are sufficient to mediate Ab-dependent phagocytosis . To explore the roles of these receptors in a simple and well-defined in vitro system, stable transfectants of fibroblasts expressing either CR3, Fc gamma RIIIB, or the combination of CR3 and Fc gamma RIIIB were generated . Cells not expressing either receptor, but exposed to the transfection protocol, were used as controls . Cell surface expression of CR3 and/or Fc gamma RIIIB were confirmed by using both flow cytometry and epifluorescence microscopy . The cell lines were analyzed for their ability to bind and internalize opsonized erythrocytes . Cells expressing both CR3 and Fc gamma RIIIB were able to both bind and phagocytose IgG-coated erythrocytes . In contrast, cells expressing CR3 only were able to phagocytose yeast, but not to bind nor phagocytose IgG-coated erythrocytes . Similarly, cells expressing Fc gamma RIIIB only were able to bind IgG-coated erythrocytes, but not to phagocytose either the erythrocytes or yeast . These studies demonstrate that, although CR3 does not participate in Ab-dependent recognition, it can complement the function of Fc gamma RIIIB to effect Ab-dependent phagocytosis . These studies also suggest that one mechanism for glycosylphosphatidylinositol-linked proteins to mediate intracellular functions is through interactions with transmembrane proteins. FEMS Microbiol Lett, 1994 Aug 15, 121(2), 207 - 15 Catabolism of 4-hydroxybenzoate in Candida parapsilosis proceeds through initial oxidative decarboxylation by a FAD-dependent 4-hydroxybenzoate 1-hydroxylase; van Berkel WJ et al.; The first two steps in the catabolism of 4-hydroxybenzoate by the ascomycetous yeast Candida parapsilosis CBS604 were investigated . In contrast to the well-known bacterial pathways and to what was previously assumed, metabolism of 4-hydroxybenzoate in C . parapsilosis proceeds through initial oxidative decarboxylation to give 1,4-dihydroxybenzene . This reaction is catalyzed by a NAD(P)H and FAD-dependent 4-hydroxybenzoate 1-hydroxylase . Further metabolism of 1,4-dihydroxybenzene to the ring-fission substrate 1,2,4-trihydroxybenzene is catalyzed by a NADPH-specific FAD-dependent aromatic hydroxylase acting on phenolic compounds . 19F-NMR experiments with cell extracts and 2-fluoro-4-hydroxybenzoate as the model compound confirm this metabolic pathway and exclude the alternative pathway proceeding through initial 3-hydroxylation followed by oxidative decarboxylation in the second step. Lancet, 1994 Aug 13, 344(8920), 444 - 5 Serodiagnosis of Penicillium marneffei infection; Yuen KY et al.; Diagnosis of Penicillium marneffei infection is often made late . We evaluated an indirect immunofluorescent antibody test for P marneffei in serum from 103 patients with persistent fever and from 78 normal subjects . Germinating conidia (initial tissue-invasion phase) and yeast-hyphae (tissue multiplication phase) forms were used as antigen . All 8 documented P marneffei cases (8%) had an IgG titre of 160 or more; the other 95 patients and all the healthy controls had an IgG titre of 40 or below . Blood culture was positive in only 1 case with HIV infection . Biopsy and culture of tissues were necessary for confirmation in the other 7 cases . The test could provide rapid presumptive diagnosis and supplement conventional culture. J Biol Chem, 1994 Aug 12, 269(32), 20771 - 9 Extent of in vivo binding by an upstream activation factor and the role of multiple binding sites in synergistic transcriptional activation; Svaren J et al.; We have analyzed site occupancy in vivo with constructs containing one or more copies of the binding site for the yeast trans-activator, yIBF . The data indicate only a modest difference in site occupancy (at most 2-fold) even though multiple copies activate transcription several hundredfold more than one copy . Using studies in which we have mutated the IES2 sequence and slightly decreased its affinity for yIBF, we have also shown that synergistic activation of transcription is dependent on overall site occupancy . Finally, synergy declines as yIBF-binding sites are separated, and loss of synergy appears to be correlated with loss of a linking surface . These results are consistent with the simultaneous contact model of synergistic activation. J Biol Chem, 1994 Aug 12, 269(32), 20340 - 6 Dominant negative analogs of NF-YA; Mantovani R et al.; NF-Y is a highly conserved heteromeric CCAAT-binding transcription factor involved in the function of several promoters . The NF-YA subunit contains a domain of high homology to yeast HAP2, which we show to be necessary and sufficient to mediate interactions with the NF-YB subunit and with DNA . Using protein affinity columns derivatized with amino acid substitution mutants, we further dissect this region into two functionally separable subdomains . The subunit association function resides in a 21-amino acid stretch, which is almost perfectly conserved among different species, while interaction with DNA resides in another short segment . We also show that DNA-binding mutants act as dominant repressors of NF-Y-DNA complex formation and of NF-Y-dependent transcription. Nucleic Acids Res, 1994 Aug 11, 22(15), 3045 - 52 The human Y4 small cytoplasmic RNA gene is controlled by upstream elements and resides on chromosome 7 with all other hY scRNA genes; Maraia RJ et al.; Ro ribonucleoproteins (RNP) constitute a class of evolutionarily conserved small cytoplasmic (sc) RNPs whose functions are unknown . In human cells four distinctive scRNAs designated hY1, hY3, hY4 and hY5 are synthesized by RNA polymerase III (pol III) and accumulate as components of Ro scRNPs . The previously isolated hY1 and hY3 genes contain upstream sequences similar to the class III promoters for U6 and 7SK snRNAs . Additional mammalian Y scRNA genes have been refractory to cloning due to interference from numerous hY-homologous pseudogenes and studies of hY RNA genes have been sparse . Although homologs of hY1 and hY3 RNAs exist in rodent cells, the smaller Y4 and Y5 RNAs do not which has allowed us to localize the hY4 scRNA gene to human chromosome 7 by assaying for its transcript in rodent X human somatic cell hybrids (SCH) . A chromosome 7-enriched yeast artificial chromosome (YAC) library was then screened and the authentic hY4 sequence was isolated by strepavidin--biotin-mediated hybrid-selection followed by poly(dA)-tailing and hemispecific PCR . The region upstream of the hY4 sequence contains a TATAAAA motif centered at -26, a candidate proximal sequence element at -63, and three octamer-like sequences located between -260 and -200 . hY4 RNA is readily detectable on Northern blots after transient transfection of the hY4 gene into mouse cells but not after transfection of a construct in which the 5' flanking region was deleted . SCHs and chromosome 7-enriched YACs were used to demonstrate that all four hY RNA genes reside on human chromosome 7. Mol Gen Genet, 1994 Aug 2, 244(3), 269 - 77 Expression of the Phytophthora infestans ipiB and ipiO genes in planta and in vitro; Pieterse CM et al.; The ipiB and ipiO genes of the potato late blight fungus Phytophthora infestans (Mont.) de Bary were isolated from a genomic library in a screen for genes induced in planta . Expression of these genes was studied during pathogenesis on various host tissues and different host plants, some of which show specific resistance against P . infestans infection . During pathogenesis on leaves and tubers of the fully susceptible potato cultivar (cv.) Ajax and on leaves of the fully susceptible tomato cv . Moneymaker, the P . infestans ipiB and ipiO genes show a transient expression pattern with highest mRNA levels in the early stages of infection . During the interaction with leaves of the partially resistant potato cv . Pimpernel, the expression is also transient but accumulation and disappearance of the mRNAs is delayed . Also in P . infestans inoculated onto a race-specific resistant potato cultivar and onto the nonhost Solanum nigrum, ipiB and ipiO mRNA is detectable during the initial stages of infection . Apparently, the expression of the ipiB and the ipiO genes is activated in compatible, incompatible and nonhost interactions . In encysted zoospores, ipiB and ipiO mRNA accumulation was not detectable, but during cyst germination and appressorium formation on an artificial surface the genes are highly expressed . Expression studies in mycelium grown in vitro revealed that during nutrient starvation the expression of the ipiB and ipiO genes is induced . For ipiO gene expression, carbon deprivation appeared to be sufficient . The ipiO gene promoters contain a sequence motif that functions as a glucose repression element in yeast and this motif might be involved in the regulation of ipiO gene expression. Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7563 - 7 Localization of a putative tumor suppressor gene by using homozygous deletions in melanomas; Weaver-Feldhaus J et al.; The p21 region of human chromosome 9 is thought to contain a gene (MLM) involved in genetic susceptibility to melanoma and a gene or genes that influence progression of certain other tumors . Genomic clones that span a large region in 9p21 surrounding the presumptive tumor suppressor gene(s) have been isolated . A set of sequence-tagged sites in this region has been developed . By using these markers and others previously reported, the 9p21 region has been studied by physical mapping in 84 melanoma cell lines . A putative tumor suppressor gene, perhaps MLM itself, has been localized to a region of less than 40 kb that lies proximal (centromeric) to the alpha-interferon gene cluster. Bioessays, 1994 Aug, 16(8), 565 - 76 Phosphatidylinositol 3-kinase; Kapeller R et al.; Currently, a central question in biology is how signals from the cell surface modulate intracellular processes . In recent years phosphoinositides have been shown to play a key role in signal transduction . Two phosphoinositide pathways have been characterized, to date . In the canonical phosphoinositide turnover pathway, activation of phosphatidylinositol-specific phospholipase C results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the generation of two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol . The 3-phosphoinositide pathway involves protein-tyrosine kinase-mediated recruitment and activation of phosphatidylinositol 3-kinase, resulting in the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate . The 3-phosphoinositides are not substrates of any known phospholipase C, are not components of the canonical phosphoinositide turnover pathway, and may themselves act as intracellular mediators . The 3-phosphoinositide pathway has been implicated in growth factor-dependent mitogenesis, membrane ruffling and glucose uptake . Furthermore the homology of the yeast vps34 with the mammalian phosphatidylinositol 3-kinase has suggested a role for this pathway in vesicular trafficking . In this review the different mechanisms employed by protein-tyrosine kinases to activate phosphatidylinositol 3-kinase, and its involvement in the signaling cascade initiated by tyrosine phosphorylation, are examined. Plant Mol Biol, 1994 Aug, 25(5), 917 - 20 Molecular cloning of a cDNA from Brassica napus L . for a homologue of acyl-CoA-binding protein; Hills MJ et al.; A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a lambda gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.) . The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly . Southern blot analysis of Brassica napus genomic DNA revealed the presence of 6 genes, 3 derived from the Brassica rapa parent and 3 from Brassica oleracea . Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots. FEBS Lett, 1994 Aug 1, 349(2), 307 - 12 Cloning and chromosomal localization of the gene coding for human protein kinase CK1; Tapia C et al.; A cDNA clone coding for human protein kinase CK1 (casein kinase 1) has been isolated and sequenced demonstrating that it corresponds to a homolog of the CK1 alpha form found in bovine brain . The derived amino acid sequence of the human CK1 alpha is identical to the bovine counterpart except that it contains 12 extra amino acids at the carboxyl end . Using this cDNA sequence and PCR amplification, YAC genomic clones that contain this human CK1 alpha sequence have been isolated . These YACs have been used for fluorescent in situ hybridization in order to localize the human CK1 alpha gene to chromosome 13q13. J Clin Invest, 1994 Aug, 94(2), 489 - 96 EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations; Giovannini M et al.; We have determined the frequency of EWS fusion transcripts in a series of primary Ewing's sarcomas and peripheral primitive neuroectodermal tumors and cells lines . Type 1 and 2 EWS-Fli1 fusions were demonstrated in 8 cell lines and 14 patient samples . Five patients with cytogenetically characterized rearrangements of chromosome 22 that did not involve chromosome 11 were included in these studies . A novel EWS-Fli1 in-frame isoform fusing EWS to exon 8 of Fli1 was isolated from a tumor with a variant t(12;22;22)(q14;p1;q12) translocation . Three in-frame isoforms of a novel hybrid transcript derived from the fusion of EWS with the ETS domain of the human erg gene were identified in patient samples and a cell line with cytogenetically unidentified or cryptic translocations involving chromosomes 21 and 22 . Interphase analysis by fluorescent in situ suppression hybridization using two overlapping erg yeast artificial chromosome clones demonstrated disruption of the erg gene on chromosome 21 in a patient sample with monosomy 22 . Our results provide new information about the involvement of EWS in small round cell tumors involving exchange of its putative RNA-binding domain with DNA-binding domains derived from different members of the ETS family of transcription factors . These studies emphasize the utility of reverse transcriptase PCR analysis and fluorescent in situ hybridization as additional diagnostic tools for differential diagnosis among small round cell tumors. Arch Biochem Biophys, 1994 Aug 1, 312(2), 340 - 8 Human pancreatic-type and nonpancreatic-type ribonucleases: a direct side-by-side comparison of their catalytic properties; Sorrentino S et al.; The catalytic properties and substrate preference of several highly purified human ribonucleases from different organs and body fluids have been examined in detail using various low-molecular-weight compounds and single- or double-stranded polyribonucleotides as substrates . All single-stranded polyribonucleotides were degraded by nonpancreatic-type (npt) RNases at a slower rate than by pancreatic-type (pt) enzymes: ptRNases were 20 times more active on RNA and poly(U) substrates and more than 6000 times more active on poly(C) . Pancreatic-type RNases degraded poly(C) faster than RNA, showing a strong preference for poly(C) over poly(U) with the following activity ratios: RNA/poly(C), 0.44; RNA/poly(U), 12; poly(C)/poly(U), 27 . In contrast, nptRNases cleaved RNA more rapidly than synthetic homopolymers, preferring poly(U) over poly(C) with the following ratios: RNA/poly(C), 130; RNA/poly(U), 10; poly(C)/poly(U), 0.08 . Human ptRNases degraded poly(A) and double-stranded polyribonucleotides about 100 and 400 times faster, respectively, than bovine RNase A . However, no measurable activity could be detected on these substrates with nptRNases . The activities of ptRNases on dinucleoside phosphates (CpN and UpN) or uridine and cytidine 2',3'-cyclic phosphates were similar to those of bovine RNase A; nptRNases, instead, cleaved only CpA and UpA at an appreciable rate . The effects of pH, ionic strength, and divalent cations on the activity of these ribonucleases were also investigated using yeast RNA as a substrate. Am J Hum Genet, 1994 Aug, 55(2), 372 - 8 A YAC contig encompassing the Treacher Collins syndrome critical region at 5q31.3-32; Dixon J et al.; Treacher Collins syndrome (TCOF1) is an autosomal dominant disorder of craniofacial development the features of which include conductive hearing loss and cleft palate . Previous studies have localized the TCOF1 locus between D5S519 (proximal) and SPARC (distal), a region of 22 centirays as estimated by radiation hybrid mapping . In the current investigation we have created a contig across the TCOF1 critical region, using YAC clones . Isolation of a novel short tandem repeat polymorphism corresponding to the end of one of the YACs has allowed us to reduce the size of the critical region to approximately 840 kb, which has been covered with three nonchimeric YACs . Restriction mapping has revealed that the region contains a high density of clustered rare-cutter restriction sites, suggesting that it may contain a number of different genes . The results of the present investigation have further allowed us to confirm that the RPS14 locus lies proximal to the critical region and can thereby be excluded from a role in the pathogenesis of TCOF1, while ANX6 lies within the TCOF1 critical region and remains a potential candidate for the mutated gene. J Virol, 1994 Aug, 68(8), 5056 - 62 Transcription stimulation of the adenovirus type 12 E1a gene in vitro by the 266-amino-acid E1A protein; Kawamura H et al.; We previously showed that the 13S but not the 12S mRNA product of the E1a gene of the highly oncogenic type 12 adenovirus (Ad12) stimulates the expression of its own gene . In this study, the mechanism for the autoregulation of the Ad12 E1a gene was investigated in vitro . The 266-amino-acid E1A protein of Ad12 was synthesized in yeast cells and purified as a 57-kDa polypeptide . The purified Ad12 E1A protein stimulated transcription from the proximal promoter of its own gene but had almost no effect on that from the distal promoter . A 35-bp upstream region including a TATA box for the proximal promoter seemed to be sufficient for transcription stimulation by the E1A protein . The Ad12 E1A protein formed a complex with a TATA box-binding protein (TBP), as does the E1A protein of nononcogenic Ad serotypes . Moreover, the E1A protein significantly reduced the binding of TBP to a TATA sequence, while it did not affect the DNA-binding activity of nuclear factor I, a stimulatory protein of the distal transcription of the Ad12 E1a gene . These results suggest that the 13S mRNA product of the Ad12 E1a gene regulates the transcription of its own gene by modulating the activity of TBP. Virology, 1994 Aug 1, 202(2), 760 - 70 The equine herpesvirus type 1 immediate-early gene product contains an acidic transcriptional activation domain; Smith RH et al.; The equine herpesvirus type 1 (EHV-1) immediate-early (IE) gene product, an ICP4 homolog, is the major regulatory protein encoded by EHV-1 during cytolytic infection . The IE gene product has been demonstrated to induce reporter gene expression directed by both homologous and heterologous viral promoters, including the EHV-1 thymidine kinase (tk) promoter, the herpes simplex virus type 1 (HSV-1) tk and ICP4 promoters, and the simian virus 40 early promoter . In this report, the transcriptional activation domain of the EHV-1 IE gene product was mapped to within an acidic, 87-amino-acid region (amino acids 3 to 89) at the amino-terminus of the IE molecule . It is demonstrated that the IE transcriptional activation domain, when fused to the DNA-binding domain of the yeast transcriptional activator GAL4, can activate gene expression in cell lines derived from at least two different species . Moreover, it is shown that the EHV-1 IR2 gene product (Harty and O'Callaghan, J . Virol . 65, 3829-3838, 1991), a truncated form of the IE polypeptide lacking IE amino acid residues 1-322 (and, therefore lacks the deduced transcriptional activation domain), fails to transactivate the EHV-1 tk promoter, but retains the ability to down-regulate the EHV-1 IE promoter . Fusion of the acidic transcriptional activation domain of the HSV-1 virion protein VP16 to the transactivation-deficient IR2 gene product restored the ability of this truncated IE polypeptide to transactivate the EHV-1 tk promoter . These findings suggest a role for the IR2 protein as a trans-repressor of EHV-1 gene expression. Virology, 1994 Aug 1, 202(2), 1058 - 60 A critical site in the cell surface receptor for ecotropic murine retroviruses required for amino acid transport but not for viral reception; Wang H et al.; The cell surface receptor (ecoR) for ecotropic host-range murine retroviruses is a Na(+)-independent transporter for cationic amino acids that is distantly related to yeast transporters for arginine, histidine, and choline . All members of this transporter family contain a conserved glutamic acid that occurs in a hydrophobic presumptive transmembrane region of ecoR at position 107 . To address the function of this site, we conservatively mutated ecoR by substituting aspartic acid at this position . The mutant protein was processed to cell surfaces where it bound the ecotropic virus envelope glycoprotein and functioned as a viral receptor, but it lacked amino acid transport activity . Functional transporter cycling is not required for viral reception by ecoR. J Acquir Immune Defic Syndr, 1994 Aug, 7(8), 799 - 806 Effect of MTP-PE liposomes and interleukin-7 on induction of antibody and cell-mediated immune responses to a recombinant HIV-envelope protein; Bui T et al.; We investigated the ability of human recombinant interleukin-7 (IL-7) to enhance the immune responses of mice