Am J Hum Genet, 1995 Sep, 57(3), 644 - 50 Assignment of the dystonia-parkinsonism syndrome locus, DYT3, to a small region within a 1.8-Mb YAC contig of Xq13.1; Haberhausen G et al.; A YAC contig was constructed of Xq13.1 in order to sublocalize the X-linked dystonia-parkinsonism (XDP) syndrome locus, DYT3 . The contig spans a region of approximately 1.8 Mb and includes loci DXS453/DXS348/IL2R gamma/GJB1/CCG1/DXS559 . For the construction of the contig, nine sequence-tagged sites and four short tandem repeat polymorphisms (STRPs) were isolated . The STRPs, designated as 4704#6 (DXS7113), 4704#7 (DXS7114), 67601 (DXS7117), and B4Pst (DXS7119) were assigned to a region flanked by DXS348 proximally and by DXS559 distally . Their order was DXS348/4704 #6/4704 #7/67601/B4Pst/DXS559 . They were applied to the analysis of allelic association and of haplotypes in 47 not-obviously-related XDP patients and in 105 Filipino male controls . The same haplotype was found at loci 67601 (DXS7117) and B4Pst (DXS7119) in 42 of 47 patients . This percentage of common haplotypes decreased at the adjacent loci . The findings, together with the previous demonstration of DXS559 being the distal flanking marker of DYT3, assign the disease locus to a small region in Xq13.1 defined by loci 67601 (DXS7117) and B4Pst (DXS7119) . The location of DYT3 was born out by the application of a newly developed likelihood method for the analysis of linkage disequilibrium. Mol Cell Biol, 1995 Sep, 15(9), 4819 - 24 The tyrosine kinase substrate p120cas binds directly to E-cadherin but not to the adenomatous polyposis coli protein or alpha-catenin; Daniel JM et al.; The tyrosine kinase substrate p120cas (CAS), which is structurally similar to the cell adhesion proteins beta-catenin and plakoglobin, was recently shown to associate with the E-cadherin-catenin cell adhesion complex . beta-catenin, plakoglobin, and CAS all have an Arm domain that consists of 10 to 13 repeats of a 42-amino-acid motif originally described in the Drosophila Armadillo protein . To determine if the association of CAS with the cadherin cell adhesion machinery is similar to that of beta-catenin and plakoglobin, we examined the CAS-cadherin-catenin interactions in a number of cell lines and in the yeast two-hybrid system . In the prostate carcinoma cell line PC3, CAS associated normally with cadherin complexes despite the specific absence of alpha-catenin in these cells . However, in the colon carcinoma cell line SW480, which has negligible E-cadherin expression, CAS did not associate with beta-catenin, plakoglobin, or alpha-catenin, suggesting that E-cadherin is the protein which bridges CAS to the rest of the complex . In addition, CAS did not associate with the adenomatous polyposis coli (APC) tumor suppressor protein in any of the cell lines analyzed . Interestingly, expression of the various CAS isoforms was quite heterogeneous in these tumor cell lines, and in the colon carcinoma cell line HCT116, which expresses normal levels of E-cadherin and the catenins, the CAS1 isoforms were completely absent . By using the yeast two-hybrid system, we confirmed the direct interaction between CAS and E-cadherin and determined that CAS Arm repeats 1 to 10 are necessary and sufficient for this interaction . Hence, like beta-catenin and plakoglobin, CAS interacts directly with E-cadherin in vivo; however, unlike beta-catenin and plakoglobin, CAS does not interact with APC or alpha-catenin. J Med Microbiol, 1995 Sep, 43(3), 224 - 9 Killing of Histoplasma capsulatum by macrophage colony stimulating factor-treated human monocyte-derived macrophages: role for reactive oxygen intermediates; Desai G et al.; The interaction of human macrophages with the yeast-form of Histoplasma capsulatum was studied . The use of culture and a short-term assay period instead of microscopy gave direct evidence of the fungicidal activity of human macrophages . The present study reports the novel finding of fungicidal activity of macrophages derived from monocytes in the presence of macrophage colony-stimulating-factor (MCSF) . The induction of fungicidal activity by this cytokine was dose dependent . MCSF at 10,000 U/ml was optimal with 73(SD3)% killing . Inhibition of macrophage killing by superoxide dismutase (SOD), but not catalase (CAT) or N-monomethyl-L-arginine (NMMA), established the role of the superoxide anion in the killing mechanism . The fungistatic activity of MCSF-derived human macrophages in a 24-h assay was also dose dependent and was not inhibited by SOD, CAT or NMMA . MCSF at 10,000 U/ml produced optimal macrophage fungistatic activity, 34.6(SD4)%. Hum Genet, 1995 Sep, 96(3), 335 - 8 Mapping of a human rRNA gene in the YAC contig surrounding the SMA candidate gene; Huschenbett J et al.; Using the yeast artificial chromosome (YAC) 116 flanking the autosomal recessive spinal muscular atrophy (SMA) gene region, we have screened a human fetal brain cDNA library and isolated the cDNA clone 14-3/9 with an insert size of 2.5 kb . The cDNA clone could be identified as part of the human rRNA gene coding for 28S rRNA with a total size of 5025 bp . The human 28S rRNA is involved in the organization of the 60S ribosomal subparticle and is arranged in a 13-kb pre-rRNA transcription unit that occurs in tandem repeat clusters . Multiple copies of the rRNA gene have been mapped by pulsed field blot hybridization in the YAC contig between YAC 66 and YAC 116, which encompasses the SMA candidate gene, and additionally in the distally localized YAC 153. Hum Genet, 1995 Sep, 96(3), 330 - 4 A recombination event occurring within two complex 5q13.1 microsatellite repeat polymorphisms suggests a telomeric mapping of spinal muscular atrophy; Yaraghi Z et al.; The gene for the childhood spinal muscular atrophies (SMAs) has been mapped to 5q13.1 . The interval containing the SMA gene has been defined by linkage analysis as 5qcen-D5S629-SMA-D5S557-5qter . We have identified a recombination event within this interval on a type-I SMA chromosome . The recombination maps to a region of multilocus microsatellite repeat (MSR) markers, and occurs between different subloci of two such markers, CMS-1 and 7613 . While the possibility of a novel mutation caused by the recombination cannot be discounted, we believe when viewed in the context of a similar recombination in a Dutch SMA family, a centromeric boundary at the recombination site for the critical SMA interval is likely . This new proximal boundary would reduce the minimal region harboring the SMA locus from approximately 1.1 Mb to approximately 600 kb. Breast Cancer Res Treat, 1995 Sep, 35(3), 233 - 41 Molecular aspects of estrogen receptor variants in breast cancer; Fuqua SA et al.; Measurements of the estrogen receptor (ER) and the estrogen-induced progesterone receptor (PgR) are used by most clinicians as indicators of both overall prognosis and likelihood of response to endocrine therapy . Patients with ER+/PgR+ tumors have the highest likelihood of response; conversely, patients with ER-/PgR-tumors have the lowest likelihood of response . Unfortunately, most patients treated successfully with endocrine therapy eventually develop endocrine-resistant disease recurrence . In an effort to study potential mechanisms of endocrine resistance, we have studied discordant ER-/PgR+ tumors, in which the normally estrogen-regulated PgR gene is induced in the apparent absence of ER . Our laboratory has previously cloned, from ER-/PgR+ tumors, a variant ER mRNA precisely missing the sequence corresponding to ER exon 5, and has demonstrated that the truncated protein product translated from this variant RNA is capable of constitutively inducing the expression of an estrogen-responsive reporter gene in a yeast expression vector system (Fuqua et al . Cancer Res 51:105-109, 1991) . In the present report we describe further experiments to characterize the activity and biological consequences of expression of this variant ER in human breast cancer cells . We have stably transfected MCF-7 human breast cancer cells with a mammalian expression vector for the exon 5 deletion variant ER . These transfected cells produce a truncated ER protein of the expected 40 kDa size . Cells expressing the exon 5 ER deletion variant constitutively express PgR, and manifest increased anchorage-independent colony formation in the absence of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS) Dev Biol, 1995 Sep, 171(1), 85 - 97 The Drosophila E93 gene from the 93F early puff displays stage- and tissue-specific regulation by 20-hydroxyecdysone; Baehrecke EH et al.; Pulses of ecdysteroids induce dramatic changes in gene expression that direct the early stages of Drosophila metamorphosis . This gene activity is reflected by the appearance of early and late puffs in the salivary gland polytene chromosomes . Curiously, the early puff genes that have been studied to date are induced by both the late larval and prepupal pulses of ecdysteroids and are expressed in many ecdysteroid target tissues, raising the question of how the hormone directs the complex stage- and tissue-specific responses associated with metamorphosis . In an effort to address this question, we have isolated and characterized the E93 gene responsible for the stage-specific 93F early puff . The E93 mRNA displays no response to ecdysteroids in late larval salivary glands but is directly induced 12 hr later by the prepupal ecdysteroid pulse, identical to the response of the 93F puff . In tissues other than the salivary gland, however, E93 displays complex spatial and temporal regulation . E93 spans at least 55 kb of genomic DNA and encodes a 146-kDa protein that has no matches in the sequence databases, but displays several characteristics of known Drosophila transcription factors . We propose that E93 acts in a stage-specific regulatory hierarchy in the salivary gland to direct its histolysis in response to the prepupal ecdysteroid pulse. Development, 1995 Sep, 121(9), 2923 - 36 Origin and specification of type II sensory neurons in Drosophila; Brewster R et al.; The peripheral nervous system (PNS) of Drosophila is a preferred model for studying the genetic basis of neurogenesis because its simple and stereotyped pattern makes it ideal for mutant analysis . Type I sensory organs, the external (bristle-type) sensory organs (es) and the internal (stretch-receptive) chordotonal organs (ch), have been postulated to derive from individual ectodermal precursor cells that undergo a stereotyped pattern of cell division . Little is known about the origin and specification of type II sensory neurons, the multiple dendritic (md) neurons . Using the flp/FRT recombinase system from yeast, we have determined that a subset of md neurons derives from es organ lineages, another subset derives from ch organ lineages and a third subset is unrelated to sensory organs . We also provide evidence that the genes, numb and cut, are both required for the proper differentiation of md neurons. Plant J, 1995 Sep, 8(3), 457 - 63 Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR; Liu YG et al.; Thermal asymmetric interlaced (TAIL-) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones . Highly specific amplification is achieved without resort to complex manipulations before or after PCR . The adaptation of this method for recovery and mapping of genomic sequences flanking T-DNA insertions in Arabidopsis thaliana is described . Insertion-specific products were amplified from 183 of 190 tested T-DNA insertion lines . Reconstruction experiments indicate that the technique can recover single-copy sequences from genomes as complex as common wheat (1.5 x 10(10) bp) . RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T-DNA transgenic lines were determined on an RFLP map . These lines, whose mapped T-DNA insertions confer hygromycin resistance, can be used for fine-scale mapping of linked phenotypic loci. Plant Mol Biol, 1995 Sep, 28(6), 1121 - 6 Isolation of a cDNA encoding a novel GTP-binding protein of Arabidopsis thaliana; Dombrowski JE et al.; A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68) . This clone is a member of a gene family that codes for a class of large GTP-binding proteins . This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins . The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein . The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels . Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron. Proc Natl Acad Sci U S A, 1995 Aug 29, 92(18), 8195 - 9 Evolutionary conservation of human TATA-binding-polypeptide-associated factors TAFII31 and TAFII80 and interactions of TAFII80 with other TAFs and with general transcription factors; Hisatake K et al.; Human transcription initiation factor TFIID is composed of the TATA-binding polypeptide (TBP) and at least 13 TBP-associated factors (TAFs) that collectively or individually are involved in activator-dependent transcription . To investigate protein-protein interactions involved in TFIID assembly and in TAF-mediated activator functions, we have cloned and expressed cDNAs encoding human TAFII80 and TAFII31 . Coimmunoprecipitation assays showed that TAFII80 interacted with TAFII250, TAFII31, TAFII20, and TBP, but not with TAFII55 . Similar assays showed that TAFII80 interacted with TFIIE alpha and with TFIIF alpha (RAP74) but not with TFIIB, TFIIE beta, or TFIIF beta (RAP30) . Further studies with TAFII80 mutations revealed three distinct interaction domains which fall within regions conserved in human TAFII80, Drosophila TAFII60, and yeast TAFII60 . The N terminus of TAFII80 (residues 1-100) interacts with both TAFII31 and TAFII20, while two C-terminal regions are involved, respectively, in interactions with TAFII250 and TFIIF alpha (RAP74) (residues 203-276) and with TBP and TFIIE alpha (residues 377-505) . The interactions between TAFII80 and general factors TFIIE alpha and TFIIF alpha (RAP74) could be important for recruitment of GTFs during activator-dependent transcription . Because TAFs 80, 31, and 20 show sequence similarities to histones H4, H3, and H2B, as well as some parallel interactions, this subset of TAFs may form a related core structure within TFIID. Nucleic Acids Res, 1995 Aug 25, 23(16), 3206 - 13 The nuclear matrix phosphoprotein p255 associates with splicing complexes as part of the {U4/U6.U5} tri-snRNP particle; Chabot B et al.; The monoclonal antibody CC3 recognizes a phosphorylated epitope present on an interphase protein of 255 kDa . Previous work has shown that p255 is localized mainly to nuclear speckles and remains associated with the nuclear matrix scaffold following extraction with non-ionic detergents, nucleases and high salt . The association of p255 with splicing complexes is suggested by the finding that mAb CC3 can inhibit in vitro splicing and immunoprecipitate pre-messenger RNA and splicing products . Small nuclear RNA immunoprecipitation assays show that p255 is a component of the U5 small nuclear ribonucleoprotein (snRNP) and the {U4/U6.U5} tri-snRNP complex . In RNase protection assays, mAb CC3 immunoprecipitates fragments containing branch site and 3' splice site sequences . As predicted for a {U4/U6.U5}-associated component, the recovery of the branch site-protected fragment requires binding of U2 snRNP and is inhibited by EDTA . p255 may correspond to the previously identified p220 protein, the mammalian analogue of the yeast PRP8 protein . Our results suggest that changes in the phosphorylation of p255 may be part of control mechanisms that interface splicing activity with nuclear organization. Cell, 1995 Aug 25, 82(4), 555 - 64 The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH; Henning KA et al.; The hereditary disease Cockayne syndrome (CS) is characterized by a complex clinical phenotype . CS cells are abnormally sensitive to ultraviolet radiation and are defective in the repair of transcriptionally active genes . The cloned CSB gene encodes a member of a protein family that includes the yeast Snf2 protein, a component of the transcriptional regulator Swi/Snf . We report the cloning of the CSA cDNA, which can encode a WD repeat protein . Mutations in the cDNA have been identified in CS-A cell lines . CSA protein interacts with CSB protein and with p44 protein, a subunit of the human RNA polymerase II transcription factor IIH . These observations suggest that the products of the CSA and CSB genes are involved in transcription. Biochem Biophys Res Commun, 1995 Aug 24, 213(3), 975 - 9 The MAP kinase cascade is not essential for transcriptional stimulation of osmolyte transporter genes; Kwon HM et al.; Kidney derived MDCK cells are protected from the stress of hypertonicity by accumulating compatible osmolytes . Accumulation of the compatible osmolytes myo-inositol and betaine is driven by hypertonicity-induced stimulation of transcription of the genes coding for the myo-inositol cotransporter and the betaine cotransporter . We tested the importance of the mitogen-activated protein kinase pathway in transcriptional activation of the genes for the two osmolytes cotransporters because this kinase pathway is rapidly activated when cells are exposed to hypertonicity and a mitogen-activated protein kinase pathway is essential for the osmo-protective transcriptional response of yeast to hypertonicity . Eliminating the activation of mitogen-activated protein kinase did not block the hypertonicity induced increase in accumulation of osmolyte transporter mRNA. Virology, 1995 Aug 20, 211(2), 593 - 7 Functional analysis of EA-D of Epstein-Barr virus; Chen LW et al.; Different mutations were generated in the diffused-form early antigen (EA-D) of Epstein-Barr virus (EBV) for study of the effects of these mutations in DNA binding, stimulating the activity of EBV-specific DNA polymerase (EBV-DP), and binding to monoclonal antibody R3 . Results revealed that the N-terminal 303 amino acids were essential for DNA binding and were sufficient for the enhancement of the activity of EBV-specific DNA polymerase . Deletion study also showed that the region recognized by the R3 monoclonal antibody was located between aa 315 and aa 377 . Our results failed to demonstrate the binding between EA-D and EBV-DP, using the proteins synthesized in vitro, suggesting that direct contact between the two proteins is not required for the EBV-DP activity in vitro . We have generated fusion between EA-D and DNA-binding domain of yeast GAL4 protein; however, this fusion protein was not able to transactivate the promoter containing UAS sequence in P3HR1 cells. Gene, 1995 Aug 19, 161(2), 243 - 8 Cloning and functional expression of the cDNA encoding rat lanosterol 14-alpha demethylase; Sloane DL et al.; Lanosterol 14 alpha-demethylase (LDM) is a cytochrome P-450 enzyme in the biosynthetic pathway of cholesterol . As such, it represents a target for cholesterol-lowering drugs . Rat LDM (rLDM) has been purified from the livers of rats treated with cholestyramine . The purified protein was used to generate tryptic fragments which were then sequenced . The amino acid (aa) sequences were used to design oligodeoxyribonucleotide primers and a DNA fragment was generated by RT-PCR to probe a phagemid library . A clone encoding rLDM was isolated from the livers of cholestyramine-treated rats . The clone contains an open reading frame encoding a polypeptide of 486 aa and a predicted molecular mass of 55 045 Da . The deduced aa sequence shows a high degree of identity to the yeast LDM sequences, as well as sequences which match typical P-450 sequence motifs . When produced in a baculovirus/insect cell culture system, LDM activity was detected and inhibited by the specific inhibitor azalanstat with an IC50 value of less than 2 nM . The isolation of this full-length coding sequence should facilitate research into understanding the direct and indirect effects of LDM in the regulation of cholesterol biosynthesis and the search for cholesterol-lowering drugs. J Biol Chem, 1995 Aug 18, 270(33), 19613 - 23 Interaction of Oct-1 with TFIIB . Implications for a novel response elicited through the proximal octamer site of the lipoprotein lipase promoter; Nakshatri H et al.; The ubiquitous human POU domain protein, Oct-1, and the related B-cell protein, Oct-2, regulate transcription from a variety of eukaryotic genes by binding to a common cis-acting octamer element, 5'-ATTTGCAT-3' . The binding of Oct-1 and Oct-2 to the functionally important lipoprotein lipase (LPL) promoter octamer site was stimulated by the general transcription factor, TFIIB . Comparative analysis of the LPL, histone H2B (H2B), and herpes simplex virus ICPO gene promoter octamer sites revealed that nucleotide sequences within and flanking the octamer sequence determined the degree of TFIIB-mediated stimulation of Oct-1 DNA binding . TFIIB was found to decrease the rate of dissociation of Oct-1 from the LPL octamer site, whereas it increased the rate of association, as well as decreased the rate of dissociation, of Oct-1 from the H2B octamer site . A monoclonal antibody against TFIIB immunoprecipitated a ternary complex containing TFIIB, Oct-1, and the LPL and H2B octamer binding sites . TFIIB did not alter the DNase I footprints generated by Oct-1 on the LPL and H2B promoters . However, Oct-1 on the TATA-binding protein and TFIIB from footprinting the perfect TATA box sequence located 5' of the LPL, NF-Y binding site . In transfection experiments, transcription from the reporters containing the LPL octamer, and either the SV40 or the yeast transcription factor GAL4-dependent enhancers, initiated at a precise position within the octamer sequence . Transcription from reporters containing the H2B octamer and the SV40 enhancer initiated at several positions within and flanking the octamer site, whereas transcription initiated at a precise position within the octamer from reporters with both the H2B octamer and the GAL4-dependent enhancer . These results suggest that octamers and their flanking sequences play an important role in positioning the site of transcription initiation, and that this could be a function of the interaction of Oct-1 with TFIIB. J Biol Chem, 1995 Aug 18, 270(33), 19545 - 50 The C terminus of mitosin is essential for its nuclear localization, centromere/kinetochore targeting, and dimerization; Zhu X et al.; Mitosin is a novel 350-kDa nuclear phosphoprotein that dramatically relocates from the evenly nuclear distribution in S phase to the centromere/kinetochore and mitotic apparatus in M phase . The dynamic relocalization of mitosin is accompanied by the phosphorylation of itself, suggesting that mitosin plays a role in mitotic progression . The molecular basis of nuclear localization and targeting of mitosin to the centromere/kinetochore were characterized using a set of epitope-tagged deletion mutants . The data indicate that the extreme C terminus (amino acids 2,487-3,113) of mitosin has both an independent centromere/kinetochore targeting domain and an unusually spaced bipartite nuclear localization signal . Moreover, the same centromere/kinetochore targeting domain was shown to be essential for the ability of mitosin to bind to itself or other putative mitosin-associated proteins through use of the yeast two-hybrid system . These results suggest that the C terminus of the mitosin is essential for its role in influencing cell cycle progression. J Biol Chem, 1995 Aug 18, 270(33), 19205 - 8 A novel transcriptional activator originating from an upstream promoter in human growth hormone gene; Labarriere N et al.; Transcription of the human growth hormone gene can start in vitro and in vivo 197 base pairs upstream from the cap site of growth hormone mRNA (Courtois, S . J., Lafontaine, D., and Rousseau, G . G . (1992) J . Biol . Chem . 267, 19736-19743) . We have now characterized the mRNA that originates from this optional promoter and have found that it occurs in human hypophysis and placenta but not in 10 other tissues . This mRNA contains an open reading frame for a protein of 107 residues that shares sequence similarity with three domains of hepatic nuclear factor-1alpha . With antibodies directed against a peptide corresponding to the C terminus of this protein, immunoreactive material was detected in a subset of cells of the adenohypophysis . When fused to the DNA-binding domain of the yeast transcription factor GAL4, the protein stimulated transcription from a GAL4-sensitive reporter gene in transiently transfected pituitary and placental cells. Science, 1995 Aug 18, 269(5226), 945 - 50 Crystal structure of a conserved protease that binds DNA: the bleomycin hydrolase, Gal6; Joshua-Tor L et al.; Bleomycin hydrolase is a cysteine protease that hydrolyzes the anticancer drug bleomycin . The homolog in yeast, Gal6, has recently been identified and found to bind DNA and to act as a repressor in the Gal4 regulatory system . The crystal structure of Gal6 at 2.2 A resolution reveals a hexameric structure with a prominent central channel . The papain-like active sites are situated within the central channel, in a manner resembling the organization of active sites in the proteasome . The Gal6 channel is lined with 60 lysine residues from the six subunits, suggesting a role in DNA binding . The carboxyl-terminal arm of Gal6 extends into the active site cleft and may serve a regulatory function . Rather than each residing in distinct, separable domains, the protease and DNA-binding activities appear structurally intertwined in the hexamer, implying a coupling of these two activities. J Biol Chem, 1995 Aug 18, 270(33), 19201 - 4 Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase; Pandey A et al.; The Eph family of receptor protein tyrosine kinases (RPTKs) is the largest family of RPTKs . The signal transduction pathways initiated by this family have only recently begun to be explored . Using a yeast two-hybrid screen to identify molecules that interact with the cytoplasmic domain of Eck, it was previously shown that activated Eck RPTK bound to and stimulated phosphatidylinositol 3-kinase (Pandey, A., Lazar, D.F., Saltiel, A . R., and Dixit, V.M . (1994) J . Biol . Chem . 269, 30154-30157) . Also isolated from this same screen was a novel protein containing SH3 and SH2 adapter modules that had striking homology to those found in the Src family of non-receptor tyrosine kinases . However, unlike other Src family members, it lacked a catalytic tyrosine kinase domain . Hence, this protein was designated SLAP for Src-like adapter protein . Using glutathione S-transferase fusion Proteins, it was demonstrated that SLAP bound to activated Eck receptor tyrosine kinase . Therefore, SLAP is a novel candidate downstream signaling intermediate and the first member of the Src family that resembles an adapter molecule. EMBO J, 1995 Aug 15, 14(16), 4102 - 7 Interchromosomal recombination is suppressed in mammalian somatic cells; Shulman MJ et al.; Homologous recombination occurs intrachromosomally as well as interchromosomally, both in mitotic (somatic) cells as well as meiotically in the germline . These different processes can serve very different purposes in maintaining the integrity of the organism and in enhancing diversity in the species . As shown here, comparison of the frequencies of intra- and interchromosomal recombination in meiotic and mitotic cells of both mouse and yeast argues that interchromosomal recombination is particularly low in mitotic cells of metazoan organisms . This result in turn suggests that the recombination machinery of metazoa might be organized to avoid the deleterious effects of homozygotization in somatic cells while still deriving the benefits of species diversification and of DNA repair. EMBO J, 1995 Aug 15, 14(16), 4073 - 82 Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication; Xie Q et al.; Geminiviruses are plant DNA viruses with small genomes whose replication, except for the viral replication protein (Rep), depends on host proteins and, in this respect, are analogous to animal DNA tumor viruses, e.g . SV40 . The mechanism by which these animal viruses create a cellular environment permissive for viral DNA replication involves the binding of a virally encoded oncoprotein, through its LXCXE motif, to the retinoblastoma protein (Rb) . We have identified such a LXCXE motif in the Rep protein of wheat dwarf geminivirus (WDV) and we show its functional importance during viral DNA replication . Using a yeast two-hybrid system we have demonstrated that WDV Rep forms stable complexes with p130Rbr2, a member of the Rb family of proteins, and single amino acid changes within the LXCXE motif abolish the ability of WDV Rep to bind to p130Rbr2 . The LXCXE motif is conserved in other members of the same geminivirus subgroup . The presence of an intact Rb binding motif is required for efficient WDV DNA replication in cultured wheat cells, strongly suggesting that one of the functions of WDV Rep may be the linking between viral and cellular DNA replication cycles . Our results point to the existence of a Rb-like protein(s) in plant cells playing regulatory roles during the cell cycle. Blood, 1995 Aug 15, 86(4), 1525 - 33 TEL and KIP1 define the smallest region of deletions on 12p13 in hematopoietic malignancies; Sato Y et al.; Unbalanced translocations as well as interstitial deletions of the short arm of chromosome 12 {del(12p)} are found as recurring chromosomal changes in a broad spectrum of hematopoietic malignancies . These changes result in the hemizygous deletion of genetic material from 12p . We mapped a yeast artificial chromosome containing the TEL gene, a cosmid contig containing part of TEL and a P1 contig containing the KIP1 gene to 12p13 . These probes were used for fluorescence in situ hybridization to analyze samples from 47 patients with various hematologic malignancies who had unbalanced translocations (25 patients) leading to loss of 12p or deletions (22 patients) involving 12p13 . The patients had acute lymphoblastic leukemia (8 cases), myelodysplastic syndrome (MDS; 11 cases), acute myeloid leukemia (AML; 10 cases), myeloproliferative disorders (4 cases), therapy-related MDS or AML (7 cases), non-Hodgkin's lymphoma (2 cases), and other hematopoietic malignancies (5 cases) . All three probes were hemizygously detected in 26 cases and were completely retained in only 9 cases . In 12 cases probes for one of the two genes were deleted, allowing us to map the smallest region of overlap of these deletions to a small genomic region that is bordered on the telomeric side by the TEL gene and on the centromeric side by KIP1 . The genomic distance between TEL and KIP1 is estimated to be about 1 to 2 Mbp. Cancer Res, 1995 Aug 15, 55(16), 3569 - 75 Farnesyltransferase inhibitors block the neurofibromatosis type I (NF1) malignant phenotype; Yan N et al.; Neurofibromatosis type I (NF1) is a hereditary tumor and developmental disorder whose defective gene was cloned previously . The protein product of the NF1 gene, neurofibromin, contains a domain that shows significant sequence homology to the known catalytic domains of mammalian Ras GTPase-activating proteins (GAP) and the yeast IRA1 and IRA2 proteins . This homologous region of neurofibromin has been shown to exhibit GAP activity toward Ras proteins . Malignant schwannoma cell lines from NF1 patients contain normal levels of GAP and nonmutated Ras proteins but barely detectable levels of neurofibromin, based on genetic mutations in the NF1 gene . Because these cells contain constitutively activated Ras.GTP, it has been proposed that neurofibromin may be the sole negative regulator of Ras in these cells . Overall, these results have implied an important role of the Ras signaling pathway in NF1 malignant schwannomas . Recently, several laboratories have developed small molecule inhibitors of Ras function that inhibit the enzyme farnesyltransferase (FT) . FT-mediated post-translational farnesylation of Ras proteins is absolutely necessary for Ras function since this modification is required for the anchoring of Ras proteins to the plasma cell membrane . Although previous studies have shown that FT inhibitors can block the growth of tumor cells carrying mutant Ras proteins, it remained unclear how this class of inhibitors would affect tumor cells such as in NF1, whose malignant growth appears to be mediated by up-regulation of wild-type Ras activity . Thus, in the current study, we investigated whether BMS-186511, a bisubstrate analogue inhibitor of FT, would inhibit the malignant growth properties of a cell line established from malignant schwannoma of an NF1 patient . Our results indicate that the malignant growth properties of ST88-14 cells, the most malignant cell line among several well-characterized NF1 cells, are inhibited by BMS-186511 in a concentration-dependent manner . Following treatment with BMS-186511, ST88-14 cells became flat, nonrefractile, were contact-inhibited, and lost their ability to grow in soft agar . In the drug-exposed cells, Ras proteins were prevented from FT-mediated membrane association . BMS-186511 was found to specifically inhibit FT, but not geranylgeranyltransferase I, a closely related enzyme . Thus, it is conceivable that FT inhibitors may ultimately become the first generation of drugs against the malignant phenotype in NF1 based on rational insights into the mechanism of action of neurofibromin. J Neurosci Res, 1995 Aug 15, 41(6), 846 - 58 GapIII, a new brain-enriched member of the GTPase-activating protein family; Baba H et al.; Ras GTPase-activating proteins (GAPs) are negative regulators of ras, which controls proliferation and differentiation in many cells . Ras GAPs have been found in a variety of species from yeast to mammals . We describe here a newly identified mammalian GAP, GapIII, which was obtained by differential screening of a rat oligodendrocyte cDNA library . GapIII putatively encodes a 834 amino acid protein with a predicted molecular weight of 96 kDa, which contains a consensus GAP-related domain (GRD) . The protein encoded by this cDNA has high homology with Gap1m, which was recently identified as a putative mammalian homolog of Drosophila Gap1 . These proteins contain three structural domains, an N-terminal calcium-dependent phospholipid binding domain, GRD, and a C-terminal PH/Btk domain . Because of the sequence homology and the structural similarities of this protein with Gap1m, we hypothesize that GapIII and Gap1m may be members of a mammalian GAP gene family, separate from p120GAP, neurofibromin (NF1), and IQGAP . To confirm the GapIII protein activity, constructs containing different GapIII-GRD domains were transformed into iral mutant yeast to determine their relative ability to replace IRA1 functionally . Constructs that contained essentially the full-length protein (all three domains), the GRD alone, or the GRD plus PH/Btk domain suppressed heat shock sensitivity of ira1, whereas constructs that contained the GRD with part of the PH/Btk domain had only a weak ability to suppress heat shock sensitivity . These results suggest that the GapIII GRD itself is sufficient to down-regulate ras proteins in yeast . Expression of GapIII mRNA (4.2 kb) was examined by Northern analysis and in situ hybridization . This mRNA was expressed at highest levels in the brain, where its expression increased with development . Lower levels of the mRNA were expressed in the spleen and lung . Among neural cells, GapIII mRNA was expressed in neurons and oligodendrocytes, but not in astrocytes . Interestingly, the expression pattern in brain is reminiscent of type 1 NF1 expression reported by Gutmann et al . (Cell Growth Differ in press, 1995) . We propose that in addition to p120GAP and neurofibromin, the GapIII/Gap1m family may be important for modulating ras activity in neurons and oligodendrocytes during normal brain development and in particular in the adult brain. J Biol Chem, 1995 Aug 11, 270(32), 19086 - 92 Junction mobility and resolution of Holliday structures by Flp site-specific recombinase . Testing partner compatibility during recombination; Lee J et al.; Absolute homology between partner substrates within the strand exchange region (spacer) is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp . Recent experiments suggest that 3-base pair homology adjacent to the points of exchange at each end of the spacer is utilized in a base complementarity-dependent strand joining reaction . Homology of the central 2 base pairs of the spacer is also critical, but how homology is tested at these two positions is unknown . We have addressed the role of homology-dependent branch migration in Flp recombination by assaying strand cleavage and resolution in a set of synthetic Holliday junctions in which the branch point is freely or partially mobile through the spacer, or is immobilized at each position within the spacer or immediately flanking it . A strong bias in the direction of Holliday resolution is observed only when the branch point is located just outside the spacer (at the junction of the Flp binding element and the spacer) . A significantly smaller bias is noticed when the branch point is frozen immediately adjacent to this position within the spacer . Resolution in these cases is most often mediated by exchange of the scissile phosphodiesters at the branch point or proximal to it, and rarely by exchange of the scissile phosphodiesters distal to it . In light of these and previous results, we discuss possible checkpoints for testing partner compatibility during Flp recombination. Genomics, 1995 Aug 10, 28(3), 420 - 8 A contiguous clone map over 3 Mb on the long arm of chromosome 11 across a balanced translocation associated with schizophrenia; Evans KL et al.; Forty-nine clones derived by microdissection of a schizophrenia-associated t(1;11)(q42.1;q14.3) breakpoint region have been assigned by somatic cell hybrid mapping to seven discrete intervals on the long arm of human chromosome 11 . Eleven of the clones were shown to map to a small region immediately distal to the translocation breakpoint on 11q . A 3-Mb contiguous clone map of this region was established by isolation of corresponding YAC recombinants . The contig was oriented and shown to traverse the translocation breakpoint by FISH and microsatellite marker analysis . This contig will facilitate the isolation of candidate sequences whose expression may be affected by the translocation. Genomics, 1995 Aug 10, 28(3), 389 - 97 The FSHD-associated repeat, D4Z4, is a member of a dispersed family of homeobox-containing repeats, subsets of which are clustered on the short arms of the acrocentric chromosomes; Lyle R et al.; Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that maps to human chromosome 4q35 . FSHD is tightly linked to a polymorphic 3.3-kb tandem repeat locus, D4Z4 . D4Z4 is a complex repeat: it contains a novel homeobox sequence and two other repetitive sequence motifs . In most sporadic FSHD cases, a specific DNA rearrangement, deletion of copies of the repeat at D4Z4, is associated with development of the disease . However, no expressed sequences from D4Z4 have been identified . We have previously shown that there are other loci similar to D4Z4 within the genome . In this paper we describe the isolation of two YAC clones that map to chromosome 14 and that contain multiple copies of a D4Z4-like repeat . Isolation of cDNA clones that map to the acrocentric chromosomes and Southern blot analysis of somatic cell hybrids show that there are similar loci on all of the acrocentric chromosomes . D4Z4 is a member of a complex repeat family, and PCR analysis of somatic cell hybrids shows an organization into distinct subfamilies . The implications of this work in relation to the molecular mechanism of FSHD pathogenesis is discussed . We propose the name 3.3-kb repeat for this family of repetitive sequence elements. Genomics, 1995 Aug 10, 28(3), 383 - 8 A YAC contig spanning the dominant retinitis pigmentosa locus (RP9) on chromosome 7p; Keen TJ et al.; The dominant retinitis pigmentosa locus RP9 has previously been localized to 7p13-p15, in the interval D7S526-D7S484 . We now report refinement of the locus to the interval D7S795-D7S484 and a YAC contig of approximately 4.8 Mb spanning this region and extending both distally and proximally from it . The contig was constructed by STS content mapping and physically orders 29 STSs in 28 YAC clones . The order of polymorphic markers in the contig is consistent with a genetic map that has been assembled using haplotype data from the CEPH pedigrees . This contig will provide a primary resource for the construction of a transcriptional map of this region and for the identification of the defective gene causing this form of adRP. Biochemistry, 1995 Aug 8, 34(31), 9840 - 50 X-ray structure of two complexes of the Y143F flavocytochrome b2 mutant crystallized in the presence of lactate or phenyl lactate; Tegoni M et al.; Flavocytochrome b2 is a flavohemo enzyme localized in the intermembrane space of yeast mitochondria, where it catalyzes the electron transfer from its substrate, L-lactate, to cytochrome c . We have obtained crystals of a flavocytochrome b2 mutant, Y143F, which are isostructural with those of the native recombinant enzyme {Tegoni, M., & Cambillau, C . (1994) Protein Sci.3, 303-314} . These crystals were grown under similar conditions to those used to obtain the recombinant enzyme, but in the presence of phenyl lactate or lactate . We report here on the structural analysis of the two complexes of flavocytochrome b2 with the reaction products at 2.9 A resolution . In both structures, the Phe143 phenyl ring keeps the same position as that of the phenolic ring of Tyr143 in both the native recombinant and in the native wild-type enzymes . The product of the reaction, phenyl pyruvate or pyruvate, is present at the active site of both subunits, and not only in subunit 2 as observed in the wild-type structure {Xia, Z.-X., & Mathews, F.S . (1990) J . Mol . Biol . 212, 837-863} . The number of interactions between the FMN and the heme domain is considerably lower in the Y143F mutant than in the native proteins . The latter finding strongly supports the hypothesis that the main role of Tyr143 in the native proteins . The latter findings strongly supports the hypothesis that the main role of Tyr143 in the native protein probably consists in establishing a hydrogen bond with the heme {Xia, Z.-X., & Mathews, F.S . (1990) J . Mol . Biol . 212, 837-863} . This interaction appears to be essential for the two domains to approach each other suitably so that the intramolecular electron transfer can occur. J Mol Biol, 1995 Aug 4, 251(1), 1 - 8 A single amino acid substitution in zinc finger 2 of Adr1p changes its binding specificity at two positions in UAS1; Cheng C et al.; The two zinc fingers of the yeast transcription factor Adr1p recognize the 6 bp sequence TTG GAG site in which the first finger, a His-X3-His finger, recognizes the G-rich triplet (GAG) and the second zinc finger, a His-X4-His finger, recognizes the T-rich sequence (TTG) . Mutations were introduced into the alpha-helical region of the second finger and the resultant mutant proteins were analyzed for DNA binding affinity and specificity in vitro and in vivo . Substituting His for Leu in the third position (+3) of the helix created a new binding specificity at two positions in the binding site . The mutant with His replacing Leu146(L146H) bound with high affinity to GGG GGG and with low affinity to TTG GGG . The single substitution at position +3 in the helix had the same effect on DNA binding specificity as substitution of the whole helix of the second finger with the helix of finger one . Changing Asp145 to Ala in the presence of His146 changed the apparent binding site of finger 2 to GT/CG . The L146H mutant zinc finger protein had the same binding specificity in vivo as in vitro . Changing the spacing between the His residues that ligand zinc in the second finger from four to three, the spacing found in the first finger of Adr1p and Zif268, did not alter the specificity or affinity of the wild-type or mutant protein. Biochem Biophys Res Commun, 1995 Aug 4, 213(1), 325 - 33 A mammalian delayed-early response gene encodes HNP36, a novel, conserved nucleolar protein; Williams JB et al.; Murine fibroblasts respond to mitogens by sequential gene expression in which immediate-early, or primary response, gene transcription factors direct expression of secondary transcripts encoded by delayed-early response (DER) genes . DER gene products include growth progression factors, but the products of several novel cDNAs are unknown . Murine and human cDNAs derived from one novel DER gene (DER12) were characterized to identify its product and to probe its role in the growth response . Both sequences encoded a hydrophobic 36 kD protein (HNP36) that was related to the yeast protein, FUN26 . Anti-murine HNP36 antibodies were prepared and shown to immunoprecipitate both mammalian in vitro translation products . Immunocytochemical staining indicated localization of HNP36 to the nucleolus where its concentration increased after mitogen stimulation . Although HNP36 protein function is unknown, its identification as a nucleolar gene transcriptionally activated by growth factors implicates it as participating in the proliferative response. J Biol Chem, 1995 Aug 4, 270(31), 18517 - 22 An abundant, trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins; Gems D et al.; A full-length mRNA encoding a secreted 26-kDa antigen of infective larvae of the ascarid nematode parasite Toxocara canis has been identified . This was characterized as a 1,082-base pair clone highly abundant (0.8-1.9%) in cDNA prepared from infective stage larvae but absent from cDNA from adult male worms . Sequence analysis revealed an open reading frame corresponding to a hydrophilic 263-amino acid residue polypeptide with a 20-residue N-terminal signal peptide, indicating that it is secreted . The 5' end of the cDNA was isolated by polymerase chain reaction using a primer containing the nematode-spliced leader sequence, SL1, showing that the mRNA is trans-spliced . The molecular mass of the putative protein with the signal peptide removed is 26.01 kDa, and antibody to the recombinant protein expressed in bacterial vectors reacts with a similarly sized protein in T . canis excretory/secretory (TES) products . An identical sequence was obtained from a genomic clone isolated by expression screening with mouse antibody to TES . The 72 amino acid residues adjacent to the signal peptide form two homologous 36-residue motifs containing 6 cysteine residues; this motif is found also in the T . canis-secreted glycoprotein TES-120 and in genes of Caenorhabditis elegans . Sequence data base searches revealed significant similarity to 7 other sequences in a newly recognized gene family of phosphatidylethanolamine-binding proteins that includes yeast, Drosophila, rat, bovine, simian, and human genes and a representative from the filarial nematode Onchocerca volvulus . Assays with the T . canis recombinant 26-kDa protein expressed as a fusion with maltose-binding protein have confirmed phosphatidylethanolamine-binding specificity for this novel product. J Biol Chem, 1995 Aug 4, 270(31), 18374 - 9 Singlet oxygen and peroxyl radicals regulate carotenoid biosynthesis in Phaffia rhodozyma; Schroeder WA et al.; Carotenoids have recently received considerable interest because of their potential in delaying or preventing degenerative diseases such as arteriosclerosis, cancer, and aging . In this study we show that the active oxygen species singlet oxygen (1O2) and peroxyl radicals differently affect carotenoid composition and biosynthesis in the yeast Phaffia rhodozyma . Photochemical generation of 1O2 with rose bengal or alpha-terthienyl induced carotenoid accumulation . In contrast, peroxyl radicals derived from t-butylhydroperoxide (tBOOH) or H2O2 decreased the content of astaxanthin and increased beta-carotene by approximately 4-fold, suggesting end product feedback regulation by astaxanthin or inhibition of biosynthetic enzymes . 14C labeling of carotenoids during oxidative stress supported the possibility of end product regulation . Carotenoids were bleached by 8 mM tBOOH within 6 h when carotenogenesis was inhibited by thymol . When treated with peroxides, a previously unreported pigment in P . rhodozyma was formed . The carotenoid had a mass of 580 Da and a molecular formula of C40H52O3 . Chemical derivatizations combined with mass and absorbance spectroscopy tentatively identified the carotenoid as dehydroflexixanthin (3,1'-dihydroxy-2,3,3',4'-tetradehydro-1',2'-dihydro-beta,psi-caro tene-4-one) . This study provides the first report of induction of astaxanthin biosynthesis by 1O2, probable feedback control by astaxanthin, and the oxidative degradation of astaxanthin to novel pigments in P . rhodozyma. Science, 1995 Aug 4, 269(5224), 690 - 3 Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase; Stoyanov B et al.; Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast . Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains . A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized . The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85 . A potential pleckstrin homology domain is located near its amino terminus . The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position. Oncogene, 1995 Aug 3, 11(3), 561 - 9 UV activation of receptor tyrosine kinase activity; Coffer PJ et al.; The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumor promoter . In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as AP-1 and NF-kappa B, as well as activation of the Src tyrosine kinase . This 'UV-response' has been well studied in mammalian cells and furthermore is conserved in yeast, however the most upstream components of this signal transduction pathway have remained elusive . Here we show that UV rapidly activates both the EGF receptor and insulin receptor, as shown by tyrosine phosphorylation of these receptors . We demonstrate that this activation is due to autophosphorylation as it only occurs in cells containing receptors with a functional kinase domain . We have further analysed the propagation of the UV-induced signal to downstream events such as, IRS-1 and Shc tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, leukotriene synthesis, MAP kinase activation and gene induction all of which are activated by UV irradiation . Importantly, we demonstrate that in cells expressing a 'kinase-dead' receptor mutant the UV-response is inhibited, blocking leukotriene synthesis, MAP kinase activation and transcriptional induction . Furthermore, prior-stimulation of cells with UV appears to reduce further responsiveness to addition of growth factor suggesting a common signaling pathway . These data demonstrate a critical role for receptor-mediated events in regulating the response mammalian cells to UV exposure. Genome Res, 1995 Aug, 5(1), 71 - 8 130 kb of DNA sequence reveals two new genes and a regional duplication distal to the human iduronate-2-sulfate sulfatase locus; Timms KM et al.; Deficiency of IDs activity results in Hunter Syndrome (mucopolysaccharidosis type II), a fatal X-linked recessive disorder . We report characterization of 28 cosmids around the IDS locus in Xq28 . Four overlapping cosmids have been sequenced in their entirety generating a 130-kb contig . These studies show the fine structure of the IDS gene and identify an IDS pseudogene-like structure located 20 kb distal to the active gene . Two novel genes have also been identified in this sequence, and one of these genes is also locally duplicated . Both homologs are expressed, and a number of alternative transcript products have been characterized . The presence of a highly conserved pseudogene-like structure within a larger duplicated region close to the IDS gene has significant implications for the study of mutations at this locus. Genome Res, 1995 Aug, 5(1), 60 - 70 Regional assignment of 68 new human gene transcripts on chromosome 11; Rosier MF et al.; We have tested 80 expressed sequence-tagged site (eSTS) markers assigned to human chromosome 11 by the Genexpress program on a panel of somatic cell hybrids containing parts of this chromosome, characterized by cytogenetic data, reference markers, and with respect to the Genethon microsatellite genetic map . Sixty-eight new gene transcripts have been assigned to 25 subregions, one of which was newly defined by five of the eSTS markers . The markers are distributed on the short and long arms in agreement with their physical length . The genic map thus obtained has been integrated with the cytogenetic, genetic, and disease maps . Two eSTS markers have been further mapped with respect to a yeast artificial chromosome (YAC) contig close to the brain-derived neurotrophic factor (BDNF) gene and thus provide potential candidate genes for the mental retardation phenotype of WAGR (Wilms' tumor, aniridia, genitourinary abnormalities and mental retardation) syndrome . Altogether, the 68 new gene transcripts localized here represent more than a threefold increase in the number of unknown regionalized genes that could reveal potential candidate genes for the numerous orphan pathologies associated with chromosome 11. Genome Res, 1995 Aug, 5(1), 5 - 12 The human obese (OB) gene: RNA expression pattern and mapping on the physical, cytogenetic, and genetic maps of chromosome 7; Green ED et al.; The recently identified mouse obese (ob) gene apparently encodes a secreted protein that may function in the signaling pathway of adipose tissue . Mutations in the mouse ob gene are associated with the early development of gross obesity . A detailed knowledge concerning the RNA expression pattern and precise genomic location of the human homolog, the OB gene, would facilitate examination of the role of this gene in the inheritance of human obesity . Northern blot analysis revealed that OB RNA is present at a high level in adipose tissue but at much lower levels in placenta and heart . OB RNA is undetectable in a wide range of other tissues . Comparative mapping of mouse and human DNA indicated that the ob gene is located within a region of mouse chromosome 6 that is homologous to a portion of human chromosome 7q . We mapped the human OB gene on a yeast artificial chromosome (YAC) contig from chromosome 7q31.3 that contains 43 clones and 19 sequence-tagged sites (STSs) . Among the 19 STSs are eight corresponding to microsatellite-type genetic markers, including seven (CA)n repeat-type Genethon markers . Because of their close physical proximity to the human OB gene, these eight genetic markers represent valuable tools for analyzing families with evidence of hereditary obesity and for investigating the possible association between OB mutations and human obesity. Mamm Genome, 1995 Aug, 6(8), 532 - 6 Human glucokinase regulatory protein (GCKR): cDNA and genomic cloning, complete primary structure, and chromosomal localization; Warner JP et al.; Null mutations in the glucokinase (GCK) gene can cause autosomal dominant type 2 diabetes (maturity onset diabetes of the young, MODY); however, MODY is genetically heterogeneous . In both liver and pancreatic islet, glucokinase is subject to inhibition by a regulatory protein (GCKR) . Given the role of GCK in MODY, GCKR is itself a candidate type 2 diabetes susceptibility gene . Here we describe the structure of full-length (2.2 kb) cDNA for human GCKR, from the hepatoblastoma cell line HepG2 . The human GCKR translation product has 625 amino acids and a predicted molecular weight of 68,700 . It has 88% amino acid identity to rat GCKR . Yeast artificial chromosomes (YAC clones) containing human GCKR were isolated, and the gene was mapped to Chromosome (Chr) 2p23 by fluorescent in situ hybridization and somatic cell hybrid analysis. Cytokine, 1995 Aug, 7(6), 483 - 90 Genomic organization of the type I and type II IL-1 receptors; Sims JE et al.; Genomic clones spanning the human type I and type II interleukin-1 receptor loci have been isolated . Approximately 75 kb of genomic DNA is required to encode the type I receptor, and approximately 38 kb to encode the type II receptor . In each case, the receptor coding region is contained in only about 20 kb of DNA, with most of the rest of each locus representing the distance between the two (type II receptor) or three (type I receptor) presumptive promoters which drive transcription . The virtually identical location of introns within the ligand-binding portion of the coding regions reinforces the presumption that the two receptors derive from a common ancestor . Yeast artificial chromosomes (YACs) have been isolated which contain the two IL-1 receptors . One of the YACs also contains the T1/ST2 IL-1 receptor-related gene . The relative map position of these three genes has been determined. Hum Reprod, 1995 Aug, 10(8), 2209 - 13 In-vitro decondensation of human spermatozoa for fluorescence in-situ hybridization; Rousseaux S et al.; In order to enhance the efficiency of fluorescence in-situ hybridization (FISH) on human interphase spermatozoa, a simple method for partial decondensation of human spermatozoa chromatin is described . The spermatozoa were washed once in 0.01 M Tris and decondensed using 10 mM dithiothreitol in 0.05 M Tris for 10-50 min, before being spread onto clean slides . Sperm samples were observed every 10 min under a phase-contrast microscope in order to modify the duration of the decondensation process . Using several centromeric or YAC probes in multicolour FISH, high hybridization rates were obtained. Mycopathologia, 1995 Aug, 131(2), 99 - 102 Prevalence of Candida ciferrii in elderly patients with trophic disorders of the legs; de Gentile L et al.; In order to define the prevalence of Candida ciferrii in onychomycosis, the fungal biota associated with toe nail onyxis was examined in 50 elderly patients with trophic disorders of the legs and in 220 patients without clinical evidence of trophic disorders . Candida ciferrii was more frequent in the first group of patients since it was recovered from 24% of these patients, whereas its prevalence was only 1.4% in the control group . Moreover, the positivity of the direct examination of toe nail scrapings, the absence of any other associated pathogens, and the repeated isolation of this yeast species for some of the patients confirmed its pathogenicity. Curr Genet, 1995 Aug, 28(3), 289 - 97 Molecular phylogeny of the fungi of the Iceman's grass clothing; Rollo F et al.; To investigate the origin of the fungal hyphae that cover the grass clothing (cloak, boots) found near the neolithic mummy known as the Tyrolean Iceman, two radiocarbon-dated samples of grass were submitted to DNA extraction . The DNA was then PCR amplified using, respectively, primers specific for the region containing the internal transcribed spacers and the 5.8s rDNA (ITS), and primers specific for an approximately 600-bp long fragment of the nuclear small-subunit ribosomal DNA (SSU rDNA) repeat units of eukaryotes . The amplification products were cloned and sequenced . Sequence analysis of 20 individual ITS clones and of ten SSU rDNA clones indicated that three types of fungal DNA can be extracted from the grass . Phylogenetic analyses, using 5.8s and SSU rDNA fungal reference sequences from EMBL and GenBank databases, suggest that the DNAs come, respectively, from a psychrophilic basidiomycetous yeast, phylogenetically close to Leucosporidium scottii, and from two ascomycetes, one of which is possibly related to the Eurotiales. Curr Genet, 1995 Aug, 28(3), 248 - 57 Characterization of peroxisome-deficient mutants of Hansenula polymorpha; Tan X et al.; In the methylotrophic yeast Hansenula polymorpha, approximately 25% of all methanol-utilization-defective (Mut-) mutants are affected in genes required for peroxisome biogenesis (PER genes) . Previously, we reported that one group of per mutants, termed Pim-, are characterized by the presence of a few small peroxisomes with the bulk of peroxisomal enzymes located in the cytosol . Here, we describe a second major group of per mutants that were observed to be devoid of any peroxisome-like structure (Per-) . In each Per- mutant, the peroxisomal methanol-pathway enzymes alcohol oxidase, catalase and dihydroxyacetone synthase were present and active but located in the cytosol . Together, the Pim- and Per- mutant collections involved mutations in 14 different PER genes . Two of the genes, PER5 and PER7, were represented by both dominant-negative and recessive alleles . Diploids resulting from crosses of dominant per strains and wild-type H . polymorpha were Mut- and harbored peroxisomes with abnormal morphology . This is the first report of dominant-negative mutations affecting peroxisome biogenesis. Protein Sci, 1995 Aug, 4(8), 1563 - 70 Characterization of leucine zipper complexes by electrospray ionization mass spectrometry; Wendt H et al.; The development of "soft" ionization methods has enabled the mass spectrometric analysis of higher-order structural features of proteins . We have applied electrospray ionization mass spectrometry (ESI-MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers . In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI-MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled-coil structures . ESI-MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4-p1 of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn) . Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI-MS as could the dimeric nature of GCN4-p1 . The formation of heteromeric two- and three-stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4-p1 and LZ, was demonstrated by ESI-MS and confirmed by fluorescence quenching experiments on fluorescein-labeled peptides . The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled-coil domains. Genome, 1995 Aug, 38(4), 817 - 23 Isolation of an 800 kb contiguous DNA fragment encompassing a 3.5-cM region of chromosome 1 in Arabidopsis using YAC clones; Vijayraghavan U et al.; The apetala1 mutation of Arabidopsis affects floral meristem identity and the development of sepal and petal primordia of the flower . We mapped the available RFLP markers on chromosome 1 that are in the general vicinity of apetala1 on a fine structure map and then chose the closest RFLP as a starting point for contiguous DNA (contig) generation . We report here a contig of about 800 kilobases (kb) that spans a 3.5 cM region of chromosome 1 . We used genomic libraries of Arabidopsis prepared in yeast artificial chromosome (YAC) vectors and the detailed characterization of 19 YACs is reported . RFLPs displayed by the end fragments from the walk were mapped to align and correlate the genetic and physical maps for this region of chromosome 1 . In this segment of the genome, 1 cM corresponds to a little over 200 kb of physical distance. Development, 1995 Aug, 121(8), 2633 - 44 Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats; Matsuno K et al.; We present a molecular and genetic analysis which elucidates the role of deltex in the Notch signaling pathway . Using the yeast 'interaction trap' assay, we define the protein regions responsible for heterotypic interactions between Deltex and the intracellular domain of Notch as well as uncover homotypic interaction among Deltex molecules . The function of the Deltex-Notch interaction domains is examined by in vivo expression studies . Taken together, data from overexpression of Deltex fragments and from studies of physical interactions between Deltex and Notch, suggest that Deltex positively regulates the Notch pathway through interactions with the Notch ankyrin repeats . Experiments involving cell cultures indicate that the Deltex-Notch interaction prevents the cytoplasmic retention of the Suppressor of Hairless protein, which otherwise is sequestered in the cytoplasm via association with the Notch ankyrin repeats and translocates to the nucleus when Notch binds to its ligand Delta . On the basis of these findings, we propose a model wherein Deltex regulates Notch activity by antagonizing the interaction between Notch and Suppressor of Hairless. Nat Genet, 1995 Aug, 10(4), 415 - 23 Gene-based sequence-tagged-sites (STSs) as the basis for a human gene map; Berry R et al.; Using our data set of 3,143 single pass sequences from human brain cDNA libraries, we have developed a strategy in which gene-based sequence-tagged-sites (STSs), derived from 3'untranslated regions of human cDNAs, are rapidly assigned to megabase-insert yeast artificial chromosomes and somatic cell hybrids to generate regional gene mapping data . Employing this approach, we have mapped 318 cDNAs, representing 308 human genes . Ninety-two of these mapped to regions implicated in human genetic diseases, identifying them as candidate genes . Extension of this strategy has the potential to result in virtually every human gene having, at its 3' end, its own associated STS, with each STS in turn specifying both a corresponding genomic clone and a specific regional location in the genome. Cancer Genet Cytogenet, 1995 Aug, 83(1), 46 - 55 Detection of 9p deletions in leukemia cell lines by interphase fluorescence in situ hybridization with YAC-derived probes; Dreyling MH et al.; Hemizygous and homozygous deletions of the type I interferon gene cluster (IFN) have been detected in about 20% of acute lymphoblastic leukemias . A putative tumor suppressor gene (TSG) is thought to be located centromeric to the IFN cluster on chromosomal bands 9p21-22 . We studied the accuracy of fluorescence in situ hybridization (FISH) for detecting deletions in interphase cells using yeast artificial chromosome (YAC) clones containing all or part of the IFN cluster . FISH probes were generated from YACs (320-1300 kb in size) by a sequence-independent amplification technique (SIA) . Fifteen cell lines (nine T-ALL, three B-cell precursor ALL, one B-ALL, one AML, one CML-BC) that had been well characterized by conventional cytogenetic analysis and molecular techniques were analyzed . We were able to detect all numerical changes of the IFN cluster including homozygous and hemizygous deletions accurately and to define subclones of the cell lines . Moreover, in six cell lines we were able to identify subclones . In dilution experiments the detection thresholds for subpopulations with homozygous and hemizygous deletions were determined to be 5% and 7.5%, respectively. Dev Biol, 1995 Aug, 170(2), 664 - 78 Expression of the novel basic helix-loop-helix gene eHAND in neural crest derivatives and extraembryonic membranes during mouse development; Cserjesi P et al.; We employed the yeast two-hybrid technique to screen a mouse embryo cDNA library for novel tissue-specific Class B basic helix-loop-helix (bHLH) transcription factors, which heterodimerize with the ubiquitously expressed Class A bHLH protein E12 . From this screen, we cloned a novel bHLH protein, which we named eHAND . Its low sequence identity with other bHLH family members and unique expression pattern during development suggest that eHAND defines a new subclass of Class B bHLH proteins . eHAND was expressed at high levels in trophoblast cells and extraembryonic membranes throughout development . The first site of eHAND expression in embryos was the heart, where it was expressed at high levels between 8.5 and 10.5 days post coitum (d.p.c.), after which transcript levels declined abruptly . By 13.5 d.p.c., eHAND expression in the heart was localized to regions of valve formation . Expression in other regions of the embryo was confined to tissues with a substantial neural crest component . eHAND was expressed in the first branchial arch and its derivatives, in the sympathoadrenal lineage, and in the enteric systems . The expression pattern of eHAND during development is distinct from that of other bHLH genes and suggests that it has a role in formation of extraembryonic tissues, heart, and neural crest derivatives. Dev Biol, 1995 Aug, 170(2), 338 - 49 Expression of the Drosophila gooseberry locus defines a subset of neuroblast lineages in the central nervous system; Buenzow DE et al.; The development of the central nervous system is known to require lineage-specific factors that are expressed in neuroblasts and their descendants, as well as molecules involved in cell-cell signaling mechanisms . The transcription factors encoded by the gooseberry locus appear to be required for the proper specification of neuroblasts and their lineages . To examine whether gooseberry expression is lineage-specific, we have utilized the FLP recombinase of yeast to positively mark cell lineages throughout Drosophila development . In this system, the actin5C promoter and the lacZ gene are separated by a polyadenylation signal flanked by two direct repeat FRTs . A heat shock is used to induce a pulse of FLP recombinase which catalyzes a site-specific recombination event between the FRT sites . The resulting excision of the polyadenylation site allows expression of lacZ from the actin5C promoter . The descendants of a cell which has undergone a recombination event are now positively marked, enabling us to compare cell lineages with the pattern of gooseberry gene expression . We find that the expression of the gooseberry locus is lineage-specific, suggesting that gooseberry may function as a selector gene in the patterning of the Drosophila central nervous system. FEBS Lett, 1995 Aug 1, 369(1), 22 - 6 From structure to function: possible biological roles of a new widespread protein family binding hydrophobic ligands and displaying a nucleotide binding site; Schoentgen F et al.; A cytosolic 21-23 kDa protein isolated from bovine brain was demonstrated to bind hydrophobic ligands, particularly phosphatidylethanolamine . The protein was encountered in numerous tissues of several species . High expression of the mRNA encoding the 21-23 kDa protein was found in rat testes . Immunohistochemical studies showed the presence of the 21-23 kDa protein in the elongated spermatids and epididymal fluid of rat testis and in brain oligodendrocytes of developing rats . As the bovine, human and rat brain 21-23 kDa proteins had only few sequence homologies with already know proteins, ti was concluded that they belong to a new protein family . In order to get additional information on the structural features of the 21-23 kDa protein, we built a molecular model which displayed a nucleotide binding site . The affinity of the bovine brain 21-23 kDa protein towards nucleotides as well as its association with cytosolic proteins and small GTP-binding proteins were demonstrated . Recently, significant sequence homologies were found with an antigen from Onchocerca volvulus, a fruit fly odorant-binding protein and the yeast protein TFS1 which is a dosage-dependent suppressor of CDC25 mutations . A positive regulation of RAS is carried out by CDC25 product which facilitates the GDP/GTP exchange on RAS proteins . These results imply that 21-23 kDa proteins function in oxidoreduction reactions and signal mechanisms during cell growth and maturation. Biochem J, 1995 Aug 1, 309 ( Pt 3), 825 - 9 Association of a RING finger protein with the cytoplasmic domain of the human type-2 tumour necrosis factor receptor; Song HY et al.; A human gene encoding a protein that specifically binds to the intracellular domain of the 75 kDa type-2 tumour necrosis factor (TNF) receptor (TNFR-2IC) has been identified using the yeast-based two-hybrid system . The N-terminal half of the TNF receptor-associated protein (TRAP) contains RING finger and zinc finger motifs often found in DNA-binding proteins including transcription factors . The 2.4 kb TRAP mRNA was barely detectable, if present at all, in lung, and variably expressed in heart, liver, placenta, brain, skeletal muscle, kidney and the pancreas; interestingly, the TRAP was more highly expressed in transformed cell lines than in normal tissues . This observation may be consistent with a role for this TRAP in promoting or regulating cellular proliferation . After in vitro transcription/translation and 35S labelling the TRAP was precipitated using a fusion protein consisting of glutathione S-transferase and the intracellular domain of TNFR-2 (TNFR-2IC), which showed that the two proteins directly interact in a mammalian cell-free system and also that identification of the TRAP was not an artifact of the two-hybrid system . By using truncated TNFR-2ICs for in vitro precipitation of 35S-TRAP, it was shown that the C-terminal half of the TNFR-2IC contains the domain necessary for interaction with TRAP . The TRAP identified in the present study shares considerable homology with, and may be the human homologue of, a mouse protein, TNF receptor-associated factor 2 (TRAF2), that binds mouse TNFR-2. Proc Natl Acad Sci U S A, 1995 Aug 1, 92(16), 7406 - 10 Physical mapping of the minimal region of loss in 5q- chromosome; Fairman J et al.; Acquired interstitial loss of all or part of the long arm of human chromosome 5 (5q-) is an anomaly that is seen frequently in patients with preleukemic myelodysplasia and acute myelogenous leukemia . Loss of a critical region of overlap at band 5q31.1 in all of these cases, with various cytogenetic breaks, signifies the existence of a key negative regulator of leukemogenesis . Previous studies have defined the proximal and distal ends of the critical region to reside between the genes for IL9 and EGR1, respectively . In this report, we describe a yeast artificial chromosome contig spanning this myeloid tumor suppressor locus . The combined order of the polymorphic loci is centromere-IL9-(D5S525-D5S558-D5S89-D5S526 -D5S393)-D5S399-D5S396-D5S414-EGR1 and telomere . The physical distance between the IL9 and EGR1 genes is estimated to be < 2.4 Mb . Here we report the utility of these polymorphic loci by detecting a submicroscopic deletion of 5q31; an acute myelogenous leukemia patient with a three-way translocation, t(5;18;17)(q31;p11;q11), as the sole anomaly revealed allele loss of the D5S399 and D5S396 loci. J Clin Invest, 1995 Aug, 96(2), 1010 - 7 Murine laminin binds to Histoplasma capsulatum . A possible mechanism of dissemination; McMahon JP et al.; Histoplasmosis, an increasingly important opportunistic infection in immunosuppressed subjects, is characterized by hematogenous dissemination of the yeast from the lung . The mechanism of this dissemination is not fully understood . Laminin, the major glycoprotein of the extracellular matrix, is known to mediate the attachment of various invasive pathogens to host tissues . In the current study, laminin is demonstrated to bind to Histoplasma capsulatum in a rapid, specific, and saturable manner . Scatchard analysis with 125I-labeled laminin revealed an estimated 3.0 x 10(4) binding sites per yeast with an apparent Kd for laminin binding of 1.6 x 10(-9) M . Laminin binding to H . capsulatum was decreased from 62 +/- 1 to 17 +/- 1 ng (P < 0.001) in the presence of 3,000 nM of Ile-Lys-Val-Ala-Val, a pentapeptide within one major cell attachment site of laminin . A 50-kD H . capsulatum laminin-binding protein was demonstrated using an 125I-Ln blot of H . capsulatum cell wall proteins . The 50-kD protein is also recognized by antibodies directed at the 67-kD laminin receptor, suggesting they are related . This study proposes a possible mechanism for H . capsulatum attachment to laminin, an important first step required for the yeast to recognize and traverse the basement membrane. Mol Cell Biol, 1995 Aug, 15(8), 4076 - 85 DNA binding specificity determinants in MADS-box transcription factors; Nurrish SJ et al.; The MADS box is a conserved sequence motif found in the DNA binding domain of a family of transcription factors which possess related but distinct DNA binding specificities . We investigated the basis of differential sequence recognition by the MADS-box proteins serum response factor (SRF), MCM1, and MEF2A, using chimeric proteins and site-directed mutants in conjunction with gel mobility shift and binding site selection assays . Deletion of sequences immediately N terminal to the SRF MADS box alters its preferred binding site to that of MEF2A, although the resulting protein still weakly binds SRF-specific sites: exclusive binding to MEF2 sites requires further mutations, at MADS-box residues 11 to 15 . In contrast to SRF, the sequence specificity of MCM1 (and of MEF2A) is determined entirely by sequences within its MADS box, and mutation of only SRF MADS-box residue 1 is sufficient to alter its binding specificity to that of MCM1 . However, changes at both MADS-box positions 1 and 11 to 15 are necessary and sufficient to alter the specificity of the MCM1 MADS box to that of MEF2, and vice versa . The role of SRF MADS-box residues which differ from those present in the other proteins was investigated by selection of functional SRF variants in yeast cells . SRF MADS-box position 1 was always a glycine in the variants, but many different sequences at the other nonconserved MADS-box residues were compatible with efficient DNA binding . We discuss potential mechanisms of DNA recognition by MADS-box proteins. Diabetes, 1995 Aug, 44(8), 999 - 1001 Identification of microsatellite markers near the human ob gene and linkage studies in NIDDM-affected sib pairs; Stirling B et al.; Non-insulin-dependent diabetes mellitus (NIDDM) is a complex metabolic disorder with a significant genetic component . Obesity is a frequent complicating factor for NIDDM . In the mouse, a number of single gene defects that result in obesity have been described . Mutations in one of these genes, the ob gene, results in both obesity and NIDDM . Recently, the cloning of the murine ob gene and its human homologue has been reported (Nature 372:425-432, 1994) . In the present study, the contribution of genetic variation at the human ob locus to NIDDM susceptibility was assessed by analyzing allele sharing in NIDDM-affected sib pairs (ASPs) for markers located near the human ob gene . Four yeast artificial chromosome clones containing the human ob gene were isolated . These clones colocalized the ob gene and two microsatellite markers, D7S514 and D7S635, to a region of 280 kb on the long arm of human chromosome 7 . The microsatellite markers were typed in 346 Mexican-American NIDDM-ASPs derived from 176 families and an additional 110 ethnically and geographically matched controls . No evidence of linkage or association between either microsatellite marker and NIDDM was observed in this population . These results suggest genetic variation in the human ob gene does not play a major role in susceptibility to NIDDM in Mexican-Americans. Hum Mol Genet, 1995 Aug, 4(8), 1355 - 64 Genetic and physical characterization of the early-onset Alzheimer's disease AD3 locus on chromosome 14q24.3; Cruts M et al.; Genetic linkage studies have provided significant evidence that a major gene defect, AD3, for familial early-onset Alzheimer's disease (EOAD) is located at chromosome 14q24.3, between the short tandem repeat (STR) markers D14S52 and D14S53 defining a genetic size of 22.7 cM for the AD3 candidate region . We constructed a physical map of the AD3 region using yeast artificial chromosomes (YACs) selected from both the CEPH and megaCEPH YAC libraries using the AD3 linked STR markers as well as new sequence-tagged sites (STSs) designed based on YAC terminal sequences . The YAC map is contiguous in the region between D14S258 and D14S53, a region of 8.2 cM, and has an estimated physical size of 4-8 Mb . The YAC contig map was used as a framework to localize three known genes, a pseudogene and two brain expressed sequence tags (ESTs) . Linkage analysis studies in two Belgian chromosome 14 EOAD families AD/A and AD/B, identified obligate recombinants in family AD/A with D14S289 and D14S61 reducing the genetic size of the candidate AD3 region substantially . The minimal AD3 candidate region measured 6.4 cM on the genetic map and is contained within six overlapping megaCEPH YACs that covered a physical distance estimated between 2 and 6 Mb . These YACs as well as other YACs in the YAC contig map are valuable resources in gene cloning efforts or genomic sequencing experiments aiming at isolating the AD3 gene. Hum Mol Genet, 1995 Aug, 4(8), 1305 - 11 Localization of 102 exons to a 2.5 Mb region involved in Down syndrome; Lucente D et al.; Exon amplification has been applied to a 2.5 Mb region of chromosome 21 that has been associated with some features of Down syndrome (DS) . Identification of the majority of genes from this region will facilitate the correlation of the over-expression of particular genes with specific phenotypes of DS . Over 100 gene fragments have been isolated from this 2.5 Mb segment . The exons have been characterized by sequence analysis, comparison with public databases and expansion to cDNA clones . Localization of the exons to chromosome 21 has been determined by hybridization to genomic Southern blots and to YAC and cosmid clones representing the region . This has resulted in a higher resolution physical map with a marker approximately every 25 kb . This integrated physical and transcript map will be valuable for fine mapping of DNA from individuals with partial aneuploidy of chromosome 21 as well as for assessing and ultimately generating a complete gene map of this segment of the genome. Biochem Mol Biol Int, 1995 Aug, 36(5), 1079 - 85 Recombinant A17 Lys human insulin: purification and characterization; Zhang BY et al.; Recombinant A17 Lys human insulin precursor expressed in yeast cells was isolated from the culture medium . After purification, the precursor was converted into A17 Lys human insulin . A17 Lys human insulin can be crystallized but has much lower receptor-binding and biological activities than porcine or human insulin where A17 is Glu . It is proposed that the A17 Glu residue may be involved in the receptor-binding or the active site of human insulin. Curr Opin Genet Dev, 1995 Aug, 5(4), 473 - 7 The role of brahma and related proteins in transcription and development; Tamkun JW; The differential transcription of Drosophila homeotic genes is maintained by the Polycomb and trithorax groups of regulatory proteins, many of which are thought to modulate chromatin structure . During the past year, studies of a trithorax group member, brahma, and related yeast and human proteins have suggested that they are components of huge complexes that assist DNA-binding regulatory proteins to overcome the repressive effects of chromatin on transcription. FEBS Lett, 1995 Aug 1, 369(1), 107 - 12 Nucleocytoplasmic transport: factors and mechanisms; Simos G et al.; In the past two years, our knowledge concerning the mechanisms of nucleocytoplasmic transport through the nuclear pore complex (NPC) has considerably expanded . The application of in vitro systems that reconstitute nuclear protein import has allowed the identification of cytosolic factors that are required for the import process . Microinjection into Xenopus oocytes and yeast genetic systems have provided interesting candidates for RNA export mediators . Functional and structural analysis of nucleoporins has demonstrated the involvement of NPC components in the transport process . Finally, new concepts have emerged such as the integration of the mechanisms of the nuclear protein import and RNA export reactions and the assembly of the transport machinery at specialised domains of the NPC. Alcohol Clin Exp Res, 1995 Aug, 19(4), 821 - 3 A strategy for the identification of candidate genes for alcohol-related phenotypes and other human disorders using rapid polymerase chain reaction mapping of gene-based sequence-tagged sites; Berry R et al.; We describe a method for the rapid identification and mapping of human genes, including those possibly contributing to disease and alcohol-related phenotypes . New human genes are identified from cDNA libraries through single-pass sequencing into the 3' untranslated (3'UT) regions of human brain cDNAs . Primers derived from the 3'UT region sequences {representing gene-based, sequence-tagged sites (STSs)} are used for polymerase chain reaction (PCR) analyses of the CEPH megabase insert yeast artificial chromosome (YAC) DNA pools . With this approach, approximately 18,000 megabase YACs can be screened and a single YAC identified using only 52 PCR reactions . The YAC localization in conjunction with other mapping techniques, such as PCR mapping to human chromosomes using somatic cell hybrids, allows identification of chromosomal band locations . In this manner, each gene can be associated with its own STS, which in turn specifies both a corresponding genomic clone and specific location in the genome . These locations can be compared with the purported locations of disease genes . The locations of the STSs can also be compared with those of Quantitative Trait Loci implicated for quantitative traits (e.g., alcohol-related phenotypes) on the basis of synteny between the mouse and human genes . Using this strategy, we found candidates for 78 human disease/syndrome genes among the first 220 genes mapped. Planta Med, 1995 Aug, 61(4), 337 - 40 Effect of peroxisomicine and related anthracenones on catalase activity; Moreno-Sepulveda M et al.; Dimeric anthracenones were isolated from toxic plants of the genus Karwinskia (Rhamnaceae) . T 514 or peroxisomicine A1 is one of these toxic compounds which produces an irreversible and selective damage on the peroxisomes of yeast cells in vivo . In this paper we now report the inhibitory effect in vitro of peroxisomicine A1 and other structurally related anthracenones on liver catalase activity . The peroxisomicine A1 produces a non-competitive inhibition with respect to H2O2 on bovine, dog, and mouse liver catalases . In the three cases Vmax was decreased whereas Km was unaffected . Other dimeric anthracenones of natural origin were also found to be inhibitors of bovine liver catalase . There is a relationship between structure and degree of inhibition of all anthracenonic compounds tested . Peroxisomicine A1 and peroxisomicine A2 caused the highest degree of inhibition (IC50 = 3.34 and 3.64 microM, respectively). Mol Gen Genet, 1995 Jul 28, 248(2), 195 - 206 Molecular genetic analysis of the ripening-inhibitor and non-ripening loci of tomato: a first step in genetic map-based cloning of fruit ripening genes; Giovannoni JJ et al.; Ripening represents a complex developmental process unique to plants . We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype . We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato, ripening-inhibitor (rin), and non-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process . A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning the rin and nor loci, respectively . To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci . Based on this analysis, the relationship in the region spanning the rin locus is estimated to be 200-300 kb/cM, while the nor locus region ratio is approximately 200 kb/1 cM . Using RFLP markers tightly linked to rin and nor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA . We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains the nor locus. Mol Gen Genet, 1995 Jul 28, 248(2), 182 - 9 Protein-protein interactions among components of the Drosophila primary sex determination signal; Liu Y et al.; Sex determination in Drosophila melanogaster is initiated in the early embryo by a signal provided by three types of genes: (1) X-linked numerator elements {e.g., sisterless-a (sis-a) and sisterless-b (sis-b)}, (2) autosomally linked denominator elements {e.g., deadpan (dpn)}, and (3) maternal factors {e.g., daughterless (da)} . This signal acts to stimulate transcription from an embryo-specific promoter of the master regulatory gene Sex-lethal (Sxl) in embryos that have two X chromosomes (females), while it fails to activate Sxl in those with only one X (males) . It has been previously proposed that competitive dimerizations among the components of this signal might provide the molecular basis for this sex specificity . Here, we use the yeast two-hybrid system to demonstrate specific protein-protein interactions among the above-mentioned factors, and to delimit their interacting domains . These results support and extend the model of the molecular basis of the X/A ratio signal. Mol Gen Genet, 1995 Jul 28, 248(2), 136 - 41 Arabidopsis heat shock factor is constitutively active in Drosophila and human cells; Hubel A et al.; Heat shock factors (HSF) are the transcriptional activators of the heat shock response . The conversion of constitutively expressed HSF to a form that can bind DNA requires the trimerization of the protein, involving leucine zipper interactions as shown for yeast, Drosophila, chicken and human HSFs . Like other metazoan HSFs, the endogenous Arabidopsis HSF displays heat shock-inducible DNA-binding activity in gel retardation assays . The heat shock-inducible binding of a recombinant Arabidopsis HSF (ATHSF1) expressed in Arabidopsis plants suggests that ATHSF1 is the major heat shock factor regulating the heat stress response . However, on transient expression in Drosophila and human cells, ATHSF1 fails to exhibit proper regulation, as demonstrated by constitutive binding to DNA, and by constitutive expression of a chloramphenicol acetyltransferase (CAT) reporter gene under the control of the Drosophila hsp70 promoter . These results suggest that the regulation of ATHSF1 is normally dependent on a specific factor that inhibits the DNA-binding and transcriptional activities under non-heat shock conditions. J Biol Chem, 1995 Jul 28, 270(30), 17858 - 65 The type I collagen pro alpha 1(I) COOH-terminal propeptide N-linked oligosaccharide . Functional analysis by site-directed mutagenesis; Lamande SR et al.; The C-propeptides of the pro alpha 1(I) and pro alpha 2(I) chains of type I collagen are each substituted with a single high-mannose N-linked oligosaccharide . Conservation of this motif among the fibrillar collagens has led to the proposal that the oligosaccharide has structural or functional importance, but a role in collagen biosynthesis has not been unambiguously defined . To examine directly the function of the pro alpha 1(I) C-propeptide N-linked oligosaccharide, the acceptor Asn residue was changed to Gln by site-directed mutagenesis . In transfected mouse Mov13 and 3T6 cells, unglycosylated mutant pro alpha 1(I) folded and assembled normally into trimeric molecules with pro alpha 2(I) . In biosynthetic pulse-chase experiments mutant pro alpha 1(I) were secreted at the same rate as wild-type chains; however, following secretion, the chains were partitioned differently between the cell layer and medium, with a greater proportion of the mutant pro alpha 1(I) being released into the medium . This distribution difference was not eliminated by the inclusion of yeast mannan indicating that the high-mannose oligosaccharide itself was not binding to the matrix or the fibroblast surface after secretion . Subtle alterations in the tertiary structure of unglycosylated C-propeptides may have decreased their affinity for a cell-surface component . Further support for a small conformational change in the mutant C-propeptides came from experiments suggesting that unglycosylated pro alpha 1(I) chains were cleaved in vitro by the purified C-proteinase slightly less efficiently than wild-type chains . Mutant and normal pro alpha 1(I) were deposited with equal efficiency into the 3T6 cell accumulated matrix, thus the reduced cleavage by C-proteinase and altered distribution in the short pulse-chase experiments were not functionally significant in this in vitro extracellular matrix model system. Science, 1995 Jul 28, 269(5223), 529 - 31 Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein; Chen JJ et al.; Human papillomaviruses (HPVs) are associated with the majority of cervical cancers and encode a transforming protein, E6, that interacts with the tumor suppressor protein p53 . Because E6 has p53-independent transforming activity, the yeast two-hybrid system was used to search for other E6-binding proteins . One such protein, E6BP, interacted with cancer-associated HPV E6 and with bovine papillomavirus type 1 (BPV-1) E6 . The transforming activity of BPV-1 E6 mutants correlated with their E6BP-binding ability . E6BP is identical to a putative calcium-binding protein, ERC-55, that appears to be localized in the endoplasmic reticulum. Biochim Biophys Acta, 1995 Jul 26, 1237(2), 162 - 8 Insertion of an uncharged polypeptide into the mitochondrial inner membrane does not require a trans-bilayer electrochemical potential: effects of positive charges; McBride HM et al.; Mitochondria with a ruptured outer membrane exhibited impaired import into this membrane of an outer membrane fusion protein containing the signal-anchor sequence of Mas70p . However, the Mas70p signal-anchor efficiently targeted and inserted the protein directly into exposed regions of the inner membrane . Import into the inner membrane was dependent on delta psi and this dependence was due to the presence of the positively-charged amino acids located at positions 2, 7, and 9 of the signal-anchor . In contrast to wild-type signal-anchor, mutants lacking the positively-charged residues mediated import into the inner membrane in both the presence and absence of delta psi . The results suggest two conclusions: (1) delta psi-dependent import of the signal-anchor sequence was due exclusively to an effect of delta psi on the positively-charged domain of the signal-anchor, rather than to an effect of delta psi on a property of the inner membrane import machinery; (2) in the absence of delta psi, the positively-charged domain of the signal-anchor prevented the otherwise import-competent signal-anchor from inserting into the membrane . This suggests that the positively-charged domain leads import across the inner membrane, and that delta psi is required to vectorially clear this domain in order to allow the distal region of the signal-anchor to enter the translocation pathway . The implications of these findings on the mechanism of import into the mitochondrial inner membrane and matrix are discussed. Nucleic Acids Res, 1995 Jul 25, 23(14), 2734 - 41 USF binds to the APB alpha sequence in the promoter of the amyloid beta-protein precursor gene; Vostrov AA et al.; The APB alpha domain in the amyloid beta-protein precursor (APP) promoter contains a nuclear factor binding domain with the core recognition sequence TCAGCT-GAC . Proteins in nuclear extracts from brain and numerous cell lines bind to this domain and it contributes approximately 10-30% to the basal APP promoter activity . Included in this domain is the CANNTG motif, which is recognized by basic helix-loop-helix transcription factors . The same motif is also present in the CDEI element of the yeast centromere and in the adenovirus major late promoter (AdMLP) . Here we present evidence based on thermostability, relative binding affinity, eletrophoretic mobility and antibody recognition that the cellular proteins that bind to the APB alpha and CDEI motifs are USF . However, the relative binding affinity for the motifs is different . The affinity of USF for AdMLP is approximately 20-fold higher than for the APB alpha sequence and 5-fold higher than for the CDEI sequence . Mutational analysis suggested that the primary determinant for USF binding affinity resides within the octamer CAGCTGAC, which is composed of the E-box consensus sequence CANNTG followed by the dinucleotide AC . The human homolog of the mouse CDEI binding protein did not bind to either the CDEI sequence or APB alpha. Nucleic Acids Res, 1995 Jul 25, 23(14), 2729 - 33 Rice-barley synteny and its application to saturation mapping of the barley Rpg1 region; Kilian A et al.; In order to facilitate the map-based cloning of the barley stem rust resistance gene Rpg1, we have demonstrated a high degree of synteny at a micro level between the telomeric region of barley chromosome 1P and rice chromosome 6 . We have also developed and applied a simple and efficient method for selecting useful probes from large insert genomic YAC and cosmid clones . The gene order within the most terminal 6.5 cM of barley chromosome 1P was compared with the most terminal 2.7 cM of rice chromosome 6 . Nine rice probes, previously mapped in rice or isolated from YAC or cosmid clones from this region, were mapped in barley . All, except one, were in synteny with the rice gene order . The exception, probe Y617R, was duplicated in barley . One copy was located on a different chromosome and the other in a non-syntenic position on barley chromosome 1P . The barley probes from this region could not be mapped to rice, but two of them were inferred to be in a syntenic location based on their position on a rice YAC . This work demonstrates the utility of applying the results of genetic and physical mapping of the small genome cereal rice to map-based cloning of interesting genes from large genome relatives. Nucleic Acids Res, 1995 Jul 25, 23(14), 2724 - 8 Conservation of fine-scale DNA marker order in the genomes of rice and the Triticeae; Dunford RP et al.; DNA markers distribute over large chromosomal regions exhibit conservation of order (collinearity) in different cereal species, but it is not known whether this is maintained on a finer scale, i.e . < or = 2 cM . To address this, sets of two or more genetically linked DNA markers were localised to yeast artificial chromosomes containing rice DNA inserts . Linkage analysis of these DNA markers in barley revealed complete correspondence with their genetic order in rice, the distance between linked sequences on rice chromosomes being < 1.6 cM or < or = 1 + 10(6) bp (1 Mb) . Thus, DNA markers separated in this range are collinear in rice, barley and, by inference, other members of the Triticeae . These results are discussed with respect to the use of rice as a key system for the isolation of cereal genes. Nucleic Acids Res, 1995 Jul 25, 23(14), 2685 - 91 A variety of DNA-binding and multimeric proteins contain the histone fold motif; Baxevanis AD et al.; The histone fold motif has previously been identified as a structural feature common to all four core histones and is involved in both histone-histone and histone-DNA interactions . Through the use of a novel motif searching method, a group of proteins containing the histone fold motif has been established . The proteins in this group are involved in a wide variety of functions related mostly to DNA metabolism . Most of these proteins engage in protein-protein or protein-DNA interactions, as do their core histone counterparts . Among these, CCAAT-specific transcription factor CBF and its yeast homologue HAP are two examples of multimeric complexes with different component subunits that contain the histone fold motif . The histone fold proteins are distantly related, with a relatively small degree of absolute sequence similarity . It is proposed that these proteins may share a similar three-dimensional conformation despite the lack of significant sequence similarity. J Biol Chem, 1995 Jul 21, 270(29), 17535 - 40 Role of acidic and phosphorylated residues in gene activation by the glucocorticoid receptor; Almlof T et al.; To investigate the role of acidic and phosphorylated amino acids in the function of the major transactivation domain (tau 1) of the glucocorticoid receptor, we have performed a mutagenesis study . Aspartic and glutamic acid residues were neutralized in clusters of 2 to 4 amino acids throughout the tau 1 domain . The activity of the mutant proteins was determined using transactivation assays in yeast and mammalian cells . Some acidic residues in the core region of tau 1 appear to play a minor role in tau 1 activity, but, generally, individual acidic residues are not critical for activity . Mutagenesis of five serine residues that are phosphorylated in the mouse glucocorticoid receptor and which are conserved in the human receptor did not affect the transactivation activity of the tau 1 domain in yeast . As in mouse cells, these serine residues are the predominant sites of phosphorylation for ectopically expressed receptor in yeast, since the mutant protein lacking all five sites had a severely reduced phosphorylation level . Mutant proteins in which larger numbers of acidic residues are neutralized show a progressive decrease in activity indicating that acidity in general is important for tau 1 function . However, our results are not consistent with the "acid blob" theory of transactivator function that has been suggested for some other activator proteins . Other putative roles for the acidity of tau 1 are discussed. J Biol Chem, 1995 Jul 21, 270(29), 17502 - 7 A peptide fragment of human DNA topoisomerase II alpha forms a stable coiled-coil structure in solution; Frere V et al.; Results are presented on a peptide fragment (1013-1056) from human DNA topoisomerase II alpha . This was selected using the procedure of Lupas et al . (Lupas, A., Van Dyke, M., and Stock, J . (1991) Science 252, 1162-1164) for its potential to adopt a stable coiled-coil structure . The same theoretical treatment rejected the segment 994-1021 proposed by Zwelling and Perry (Zwelling, L . A., and Perry, W . M . (1989) Mol . Endocrinol . 3, 603-604) as a possible core for leucine-zipper formation . Our experimental studies combine cross-linking and CD analysis . Cross-linking establishes that the 1013-1056 fragment forms a stable homodimer in solution . Effects of increasing peptide concentration on CD spectra confirm that only the 1013-1056 fragment can undergo a coiled-coil stabilization from an isolated alpha-helix . Unfolding experiments further show that the coiled-coil is more stable in guanidium chloride than in urea . Values of -6.8 and -7.4 kcal/mol for the dimerization free energy are determined by thermal and urea unfolding, respectively . These are strikingly similar to the value recently found for the dissociation/reassociation of the entire yeast topoisomerase II from sedimentation equilibrium experiments (Lamhasni, S., Larsen, A . K., Barray, M., Monnot, M., Delain, E., and Fermandjian, S . (1995) Biochemistry 34, 3632-3639), although their significance relatively to topoisomerase II undoubtedly requires further analysis. J Biol Chem, 1995 Jul 21, 270(29), 17159 - 70 Functional dissection of P-glycoprotein nucleotide-binding domains in chimeric and mutant proteins . Modulation of drug resistance profiles; Beaudet L et al.; We wished to determine if the two nucleotide-binding domains (NBD) of P-glycoprotein are functionally equivalent and interchangeable, and if not, which segments and amino acids are important for proper function of each NBD within the context of the C- or N-terminal P-glycoprotien halves . For this, we constructed and tested the biological activity in yeast and mammalian cells of a series of chimeric mdr3 cDNAs in which discrete domains of the N-terminal NBD (NBD1) were replaced by the homologous segments of the C-terminal NBD (NBD2) . Although most NBD1 segments could be replaced without loss of P-glycoprotein function, exchange of small segments near the Walker B motif caused a dramatic reduction in Adriamycin, actinomycin D, and colchicine resistance in LR73 cells, as well as in FK506 resistance and STE6 complementation in yeast . Site-directed mutagenesis identified amino acid positions 522-525 (ERGA-->DKGT) and 578 (Thr-->Cys) as essential for proper function of NBD1 in the context of the N-terminal half P-glycoprotein . In addition, the observed phenotype of the mutants (altered drug resistance profile) suggests that these residues may participate directly or indirectly in substrate interactions and are possibly implicated in signal transduction from NBDs to transmembrane domains, the primary sites of drug binding in P-glycoprotein. Genomics, 1995 Jul 20, 28(2), 315 - 27 Rapid construction of integrated maps using inner product mapping: YAC coverage of human chromosome 11; Perlin MW et al.; Inner product mapping (IPM) has been proposed as a hybridization-based method for achieving low-cost, high-throughput, high-resolution radiation hybrid (RH) mapping of clones . Using Alu-PCR products of chromosome 11-specific clones, we serially hybridized a set of RHs against gridded filters of YACs having an average size of 350 kb . We then combined these hybridization data with preexisting RH map data to build an inner product map . This binning of 865 YACs provides the first high-resolution large-scale (> twofold redundancy) clonal coverage of human chromosome 11 and is the first inner product map ever constructed . We verified the accuracy and precision of this chromosome 11 map by performing a novel likelihood analysis on independent YAC hybridization data . These results establish that IPM is a highly rapid, inexpensive, accurate, and precise large-scale long-range mapping method, particularly when preexisting RH maps are available, and that IPM can replace or complement more conventional short-range mapping methods . IPM may enable the rapid construction of sequence-ready maps and the binning of expressed sequences. Genomics, 1995 Jul 20, 28(2), 147 - 53 Construction of 110 cosmid markers and a 4.5-Mb YAC contig on human chromosome 8p12-q11; Kurimasa A et al.; Microcell hybrids containing various regions of human chromosome 8 were formed by microcell-mediated transfer of neo-tagged chromosome 8 into the cells derived from severe combined immunodeficiency (SCID) mouse . Thus, 110 cosmid markers were isolated from SV40-transformed SCID fibroblast cell line (SCVA) containing a p12-q11.1 region of human chromosome 8 and were assigned to eight regions in 8p12-q11.1, using a microcell-hybrid panel . For positional cloning of a human gene that restores the DNA-repair defect in a mouse with SCID on 8p11.1-q11.1 (SCID region), we constructed a yeast artificial chromosome (YAC) contig of about 4.5 Mb . Overlapping YACs were further aligned by restriction mapping, using rare-cutting restriction endonucleases . The cosmids and YAC contig should facilitate isolation of the SCID gene and other genes, such as the Werner syndrome-responsible gene in or near this region. Oncogene, 1995 Jul 20, 11(2), 397 - 403 The Ras suppressor RSU-1 localizes to 10p13 and its expression in the U251 glioblastoma cell line correlates with a decrease in growth rate and tumorigenic potential; Tsuda T et al.; Rsu-1, which was isolated based on its ability to suppress transformation by v-Ras, is a highly conserved gene which shares homology with yeast adenylyl cyclase in the region required for activation by Ras . Genomic DNA clones of human RSU-1 have been isolated and used as a probe for fluorescence in situ hybridization (FISH) to assign RSU-1 to 10p13, confirming the previous results of somatic cell hybrid mapping localizing RSU-1 to chromosome 10 . Screening of more than 20 human tumor cell lines for RSU-1 expression revealed that most cell lines contained abundant RSU-1 RNA and protein . However, the p33 RSU-1 protein was undetectable in the U251 glioblastoma cell line and transfection of a rsu-1 expression vector into U251 cells yielded a cell line in which rsu-1 was under the control of a regulatable metallothionein promoter . Addition of Cd2+ to the U251-Rsu-1 transfectant resulted in transcription of rsu-1 RNA and the accumulation of p33 Rsu-1 protein . Appearance of the Rsu-1 protein correlated with a reduction in growth rate of the U251-Rsu-1 transfectant . In addition, reduction in anchorage independent growth and phenotypic alteration in U251-Rsu-1 transfectant agar colonies was observed . Two U251-Rsu-1 transfectant cell lines were non tumorigenic when injected subcutaneously into athymic nude mice . These results, in conjunction with the frequent deletions observed in chromosome 10 in glioblastomas, suggest that RSU-1 loss of function may play a role in the progression of this disease. Oncogene, 1995 Jul 20, 11(2), 239 - 44 Induction of neurite outgrowth by MAP kinase in PC12 cells; Fukuda M et al.; Treatment of PC12 cells with nerve growth factor (NGF) results in neural differentiation of the cells, inducing neurite outgrowth . Ras protein has been shown to play an essential role in this process . To examine whether or not the MAP kinase (MAPK) cascade mediates the NGF- and Ras-induced neural differentiation process, we injected PC12 cells with constitutive active forms of each components of the MAPK cascade . When a moderately active mutant of Xenopus MAPK kinase (S222E-MAPKK) in which Ser 222 was changed into glutamic acid was injected, the neurite outgrowth of PC12 cells occurred to some extent . Injection of an N-terminal truncated STE11 protein (delta N-STE11), a constitutively active form of STE11 which is a yeast MAPKK kinase, induced neurite outgrowth in PC12 cells . Furthermore, injection of thiophosphorylated MAPK, but not purified active MAPK, into PC12 cells resulted in neurite outgrowth . Thiophosphorylated MAPK was resistant to protein phosphatase 2A treatment, while purified active MAPK was inactivated by this treatment . All these results have suggested that sustained activation of MAPK is sufficient for PC12 cell differentiation . In accord with this, the delta N-STE11- or S222E- MAPKK-induced neurite outgrowth was inhibited by coinjection of CL-100 protein, a dual-specificity phosphatase that is capable of inactivating MAPK. Biochim Biophys Acta, 1995 Jul 19, 1250(2), 144 - 8 Purification and characterization of alpha-D-mannosidase from Aspergillus sp; Gaikwad SM et al.; alpha-D-Mannosidase from Aspergillus sp . was purified to homogeneity by preparative polyacrylamide gel