FEBS Lett, 2000 Feb 11, 467(2-3), 273 - 8 Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants; Sohlenkamp C et al.; We have cloned and characterized the first member of a novel family of ammonium transporters in plants: AtAMT2 from Arabidopsis thaliana . AtAMT2 is more closely related to bacterial ammonium transporters than to plant transporters of the AMT1 family . The protein was expressed and functionally characterized in yeast . AtAMT2 transported ammonium in an energy-dependent manner . In contrast to transporters of the AMT1 family, however, AtAMT2 did not transport the ammonium analogue, methylammonium . AtAMT2 was expressed more highly in shoots than roots and was subject to nitrogen regulation. Am J Trop Med Hyg, 1999 Dec, 61(6), 914 - 9 Environmental conditions favoring bat infection with Histoplasma capsulatum in Mexican shelters; Taylor ML et al.; Histoplasma capsulatum was isolated from gut, lung, liver, and spleen of 17 of 208 captured bats belonging to 6 different genera and species . Three of the 17 infected bats were from the State of Guerrero and 14 were from the State of Morelos . All were adult bats: 6 males (1 Pteronotus parnellii, 2 Natalus stramineus, 2 Artibeus hirsutus, and 1 Leptonycteris nivalis) and 11 females (1 Myotis californicus, 1 Mormoops megalophylla, 8 A . hirsutus, and 1 L . nivalis) . High rates of bat infection with H . capsulatum were found in the monitored sites of the State of Morelos . Histoplasma infection of N . stramineus, A . hirsutus, and L . nivalis should be considered as the first records in the world . The fungus isolated from infected bats was identified by its typical mycelial-phase morphology and by its yeast-phase conversion . Exoantigen production confirmed the fungal identification by the presence of specific precipitation lines in double immunodiffusion assays using human immune serum . Histopathologic studies showed intracellular yeast-like cells compatible with H . capsulatum yeast-phase in tissues of several bats, especially in pulmonary (intra-alveolar and septal) macrophages, with none or minimal tissue reaction . In contrast to past reports, present data support a high risk of bat infection with H . capsulatum in Mexican cave environments. Mol Endocrinol, 2000 Feb, 14(2), 295 - 306 Association of 2',5'-oligoadenylate synthetase with the prolactin (PRL) receptor: alteration in PRL-inducible stat1 (signal transducer and activator of transcription 1) signaling to the IRF-1 (interferon-regulatory factor 1) promoter; McAveney KM et al.; The PRL receptor (PRL-R) signals through the Janus tyrosine kinases (JAK) and other non-JAK tyrosine kinases, some of which are preassociated with the PRL-R . To clone PRL-R interacting proteins, the intracellular domain (ICD) of the long form of the PRL-R was used in a yeast two-hybrid screen of a human B cell cDNA library . One PRL-R interacting protein was identified as the 42-kDa form of the enzyme 2',5'-oligoadenylate synthetase (OAS) . The in vivo interactions in yeast were further confirmed by an in vitro interaction assay and by coimmunoprecipitation in transfected mammalian cells . Functionally, OAS reduced the basal activity of two types of promoters in transiently transfected COS-1 cells . In the presence of PRL, OAS inhibited PRL induction of the immediate early IRF-1 (interferon-regulatory factor 1) promoter, but not PRL induction of the differentiation-specific beta-casein promoter, suggesting that OAS exerts specific effects on immediate early gene promoters . The inhibitory effects of OAS were accompanied by a reduction in PRL-inducible Stat1 (signal transducer and activator of transcription 1) DNA binding activity at the IRF-1 GAS (interferon-gamma-activated sequence) element . These results demonstrate a novel interaction of OAS with the PRL-R and suggest a role for OAS in modulating Stat1-mediated signaling to an immediate early gene promoter . Although previously characterized as a regulator of ribonuclease (RNase) L antiviral responses, OAS may have additional effects on cytokine receptor signal transduction pathways. Ann Genet, 1999, 42(4), 202 - 9 Double telomeric signals on single chromatids revealed by FISH and PRINS; Philippe C et al.; FISH probes for all human telomeres and specific telomeric probes that hybridize to unique sequences on individual chromosomes have been used to characterize the telomeric hybridization pattern of human peripheral blood lymphocytes and bone-marrow cells in interphase and metaphase chromosomes . We have identified the existence of double hybridization signals on chromatids both with the (TTAGGG)n telomere repeat arrays and on non chromosome-specific subtelomeric regions as well as on chromosome-specific sequences located several kilobases from the end of chromosomes . Preliminary results using cosmid or YAC probes that hybridize to regions rich in GC sequences also revealed double fluorescent spots on a single chromatid . Double spots were detected by PRINS on terminal and interstitial telomeric sequences in avian cells . The significance of this phenomenon is discussed based on some models of chromatid and DNA organization such as uninemy, looped chromatid organization and quartet DNA structures . The occurrence of double spots should be taken into consideration for the clinical cytogenetic diagnosis of duplications. Oral Dis, 2000 Jan, 6(1), 3 - 11 Isolation and identification of Candida from the oral cavity; Williams DW et al.; A number of methods of sampling the oral cavity for the presence of candida have been developed . Such techniques play an important role in the diagnosis and management of oral candidosis . In the past, identification of candida isolated from the oral cavity has usually been limited to the genus Candida or to the species C . albicans . However, with the recognition that Candida species differ in the production of putative virulence factors and sensitivity to antifungal agents, greater emphasis has been placed on identification of isolates to species level . As a result a range of commercially available systems for yeast identification can now be used in conjunction with traditional identification procedures. Biochem Biophys Res Commun, 2000 Jan 27, 267(3), 906 - 11 BERP, a novel ring finger protein, binds to alpha-actinin-4; El-Husseini AE et al.; We recently identified BERP as a novel RING finger protein belonging to the RBCC protein family . It contains an N-terminal RING finger, followed by a B-box zinc finger and a coiled-coil domain . BERP interacts with the tail domain of the class V myosins through a beta-propeller structure in the BERP C-terminal . To identify other proteins interacting with BERP, the yeast two-hybrid strategy was employed, using the RBCC domain as bait . Screening of a rat brain cDNA library identified alpha-actinin-4 as a specific binding partner for the N-terminus of BERP . This actinin isoform could be immunoprecipitated together with BERP from HEK 293 cells transfected with expression constructs for BERP and alpha-actinin-4 . These proteins could also be colocalized immunohistochemically in the cytoplasm of differentiated PC12 cells . We suggest that BERP may anchor class V myosins to particular cell domains via its interaction with alpha-actinin-4 . Genomics, 2000 Jan 15, 63(2), 294 - 7 Structure, expression, and chromosomal localization of the mouse Atox1 gene; Hamza I et al.; Copper trafficking in eukaryotes involves small proteins termed metallochaperones, which mediate copper delivery to specific intracellular sites . Previous studies in yeast and human cell lines have suggested that Atox1 plays a critical role in copper delivery to the secretory pathway . In the present study, a mouse Atox1 (mAtox1) cDNA was cloned and shown to encode an open reading frame with 85% amino acid identity to human Atox1 . RNA blot analysis revealed that mAtox1 was expressed as a single transcript in multiple tissues, and immunoblotting indicated that the relative abundance of mAtox1 mRNA directly correlated with mAtox1 protein . Analysis of the mAtox1 gene locus revealed a genomic structure with four exons encompassing a total of 14.5 kb . RFLP and haplotype analyses indicated that the mAtox1 locus was tightly linked to the Trhr and D15Bir7 loci on mouse chromosome 15 . Taken together, these data reveal marked evolutionary conservation of Atox1 structure and provide a genomic organization and localization that will aid in the genetic deciphering of the molecular role of this protein in copper homeostasis . Genomics, 2000 Jan 15, 63(2), 271 - 8 Genetic refinement and physical mapping of the CMT4B gene on chromosome 11q22; Bolino A et al.; Charcot-Marie-Tooth disease type 4B (CMT4B) is a demyelinating autosomal recessive motor and sensory neuropathy characterized by focally folded myelin sheaths in the peripheral nerve . We recently mapped the CMT4B gene to a 5-cM interval on chromosome 11q22, using homozygosity mapping and haplotype sharing analysis on a large inbred pedigree . In the present study, we report the construction of a YAC-based transcript map across the 5-cM critical region, including 26 YACs, 35 STSs, and 52 ESTs . Furthermore, using 15 additional physically ordered microsatellite markers from the 11q22 region on the original inbred family, we were able to narrow the critical interval for the gene to 2 Mb, which is now flanked by markers D11S1757 and CHLC-GATA3B05 . Finally, after computer analysis of the 33 ESTs assigned to the 2-Mb interval, we demonstrated that 21 different transcripts as well as 3 known genes might represent potential candidates for the disease . Bioorg Med Chem Lett, 2000 Jan 17, 10(2), 189 - 92 Synthesis of dolichyl phosphate derivatives with fluorescent label at the omega-end of the chain, new tools to study protein glycosylation; Shibaev VN et al.; Derivatives of dolichyl phosphate (Dol-P) with 2-aminopyridine or 1-aminonaphtalene fluorophore groups at the omega-end of the chain were synthesized . These products serve as substrates for recombinant yeast Dol-P-mannose synthase . Fluorescence resonance energy transfer between a Trp residue of the enzyme and the 1-aminonaphtalene group of the Dol-P analogue was demonstrated. Biochim Biophys Acta, 1999 Dec 23, 1489(2-3), 452 - 6 Cloning, expression and characterization of a novel human Ras-related protein that is regulated by glucocorticoid hormone; Tu Y et al.; Ras proteins are a family of guanine nucleotide (GDP and GTP)-binding proteins that play central roles in essential signal transduction pathways . We have isolated in a yeast two-hybrid screen a human cDNA encoding a new protein that is highly homologous (98% identical at the protein level) to mouse DexRas1, a member of the Ras superfamily . The human DexRas1 is expressed in a variety of tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas, with the strongest expression in the heart . Using human fibrosarcoma HT-1080 cells as a model system, we show that the expression of human DexRas1 is stimulated by dexamethasone, suggesting a role of human DexRas1 in dexamethasone-induced alterations in cell morphology, growth and cell-extracellular matrix interactions. Biochim Biophys Acta, 1999 Dec 23, 1489(2-3), 303 - 14 Identification of novel isoforms of human RAD52; Kito K et al.; In yeast, RAD52 has been shown to be essential for homologous recombination of DNA and to be involved in the repair of double-stranded DNA breaks . Recently, the human homologue of yeast RAD52, a 418-amino-acid protein, has been identified . In this study, we report three different isoforms of human RAD52 isolated from brain and testis cDNA libraries . cDNAs of these isoforms contain distinct insertions and encode truncated proteins due to translational frame-shifts . The three isoforms consist of 226-, 139-, and 118-amino-acid residues, and are designated as RAD52beta, gamma, and delta, respectively . The original RAD52 is termed as RAD52alpha in this paper . Messages of these isoforms have been detected in various human tissues . We found that the RAD52 isoforms were unable to interact with RAD52alpha because of partial defect of the self-interaction domain . Furthermore, like RAD52alpha, the isoforms have been shown to bind to both single-stranded and double-stranded DNA . These results suggest that RAD52beta, gamma, and delta might affect RAD52alpha function through their DNA-binding property and their inability to bind to RAD52alpha . Thus, these isoforms might act as dominant negative mutants or negative regulators of RAD52alpha. Mol Cells, 1999 Dec 31, 9(6), 631 - 7 The SH2-SH2-SH3 domain of phospholipase C-gamma1 directly binds to translational elongation factor-1alpha; Kim MJ et al.; Phospholipase C-gamma1 (PLC-gamma1) is a lipase that hydrolyzes PIP2 to generate two second messengers, IP3 and DAG . By using the yeast two-hybrid system, we identified the translational elongation factor-1alpha (EF-1alpha) as a binding protein of PLC-gamma1 from the human B-lymphocyte library . Direct interaction between EF-1alpha and PLC-gamma1 was confirmed by the in vitro binding experiment using purified PLC-gamma1 . Furthermore, from the in vitro binding experiment, we could demonstrate that the carboxyl terminal region of EF-1alpha is involved in the interaction with PLC-gamma1, and that both SH2 and SH3 domains of PLC-gamma1 are required for the interaction with EF-1alpha . In vivo interaction between EF-1alpha and PLC-gamma1 was confirmed by the immunoprecipitation experiment using anti-EF-1alpha antibody . The interaction between EF-1alpha and PLC-gamma1 was enhanced by EGF-treatment . Taken together, we suggest that EF-1alpha might play a role in PLC-gamma1-mediated signal transduction. Curr Genet, 2000 Jan, 37(1), 65 - 73 Cytochrome P450 oxidoreductase gene and its differentially terminated cDNAs from the white rot fungus Phanerochaete chrysosporium; Yadav JS et al.; The white rot fungus Phanerochaete chrysosporium metabolizes a range of xenobiotics via P450 mono-oxygenation, particularly under peroxidase-suppressing culture conditions . Here we report the cloning and analysis of the gene from this fungus for the cytochrome P450 oxidoreductase (CPR) and its differentially terminated cDNAs . Using a PCR-based approach with degenerate primers, a 285-bp genomic fragment was isolated from the two widely studied strains BKM-F 1767 and ME 446, and was identified as a CPR gene segment based on sequence comparison with the database . A clone containing the full-length CPR gene was isolated from a BKM-F 1767 genomic library using the PCR-generated segment as a probe, and the 3937-bp insert was sequenced by gene walking . Based on the detection of conserved CPR motifs, a coding region of 2381 bp was identified with a 991-bp segment 5' to the putative ATG start codon . Two cDNAs with differentially terminated transcripts were isolated and sequenced . Comparison of the gene and the cDNA sequences confirmed the presence of three introns (62 bp, 50 bp, and 58 bp) . Sequence identity and a phylogenetic comparison of the deduced protein (736 aa) with other CPRs in the database suggested that P . chrysosporium CPR is the largest CPR known and is more closely related to animal (36-38%) and yeast (37-38%) CPRs than to plant CPRs (33-35%) . The availability of this gene will facilitate further studies on understanding the potent xenobiotic mono-oxygenation systems in this model white rot fungus. Eur J Biochem, 2000 Feb, 267(4), 1019 - 29 Specific inhibition of barley alpha-amylase 2 by barley alpha-amylase/subtilisin inhibitor depends on charge interactions and can be conferred to isozyme 1 by mutation; Rodenburg KW et al.; alpha-Amylase 2 (AMY2) and alpha-amylase/subtilisin inhibitor (BASI) from barley bind with Ki = 0.22 nM . AMY2 is a (beta/alpha)8-barrel enzyme and the segment Leu116-Phe143 in domain B (Val89-Ile152), protruding at beta-strand 3 of the (beta/alpha)8-barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2 . In the AMY2-BASI crystal structure {F . Vallee, A . Kadziola, Y . Bourne, M . Juy, K . W . Rodenburg, B . Svensson & R . Haser (1998) Structure 6, 649-659} Arg128AMY2 forms a hydrogen bond with Ser77BASI, while Asp142AMY2 makes a salt-bridge with Lys140BASI . These two enzyme residues are substituted by glutamine and asparagine, respectively, to assess their contribution in binding of the inhibitor . These mutations were performed in the well-expressed, inhibitor-sensitive hybrid barley alpha-amylase 1 (AMY1)-(1-90)/AMY2-(90-403) with Ki = 0.33 nM, because of poor production of AMY2 in yeast . In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T129R/K130P in the inhibitor-insensitive hybrid AMY1-(1-161)/AMY2-(161-403) . The binding energy was reduced by 2.7-3.0 kcal.mol-1 as determined from Ki after the mutations R128Q and D142N . This corresponds to loss of a charged interaction between the protein molecules . In contrast, sensitivity to the inhibitor was gained (Ki = 7 microM) by the mutation T129R/K130P in the insensitive isozyme hybrid . Charge screening raised Ki 14-20-fold for this latter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants . Thus electrostatic stabilization was effectively introduced and lost in the different mutant enzyme-inhibitor complexes and rational engineering using an inhibitor recognition motif to confer binding to the inhibitor mimicking the natural AMY2-BASI complex. Oncol Rep, 2000 Mar-Apr, 7(2), 381 - 5 p53 inactivating mutations in chinese breast carcinomas; Gong ZY et al.; While previous reports on breast cancers indicate that Caucasian women have a low frequency of p53 mutations, higher frequencies of mutations are reported in some Japanese populations . There are few reports regarding p53 mutations in Chinese breast carcinomas . Using a previously established sensitive p53 yeast functional assay, we screened 23 Chinese breast carcinomas for mutations . The p53 was mutated in 5/23 (21.7%) specimens . Two of these mutations were detected in exon 5 and one was detected in each of exons 6, 7 and 8 . All of these mutations have previously been shown to be mutated in Caucasian and Japanese breast cancers, but two have not previously been observed in Chinese breast cancers and one has also not been observed in Japanese. J Biol Chem, 2000 Feb 18, 275(7), 5222 - 7 R-Ras contains a proline-rich site that binds to SH3 domains and is required for integrin activation by R-Ras; Wang B et al.; R-Ras contains a proline-rich motif that resembles SH3 domain-binding sites but that has escaped notice previously . We show here that this site in R-Ras is capable of binding SH3 domains and that the SH3 domain binding may be important for R-Ras function . A fusion protein containing the SH3 domains of the adaptor protein Nck interacted strongly with the R-Ras proline-rich sequence and with the intact protein . The binding was independent of whether R-Ras was in its GDP or GTP form . The Nck binding, which was mediated by the second of the three SH3 domains of Nck, was obliterated by mutations in the proline-rich sequence of R-Ras . The interaction of Nck with R-Ras could also be shown in yeast two-hybrid assays and by co-immunoprecipitation in human cells transfected with Nck and R-Ras . Previous results have shown that the expression of a constitutively active R-Ras mutant, R-Ras(38V), converts mouse 32D monocytic cells into highly adherent cells . Introducing the proline mutations into R-Ras(38V) suppressed the effect of R-Ras on 32D cell adhesion while not affecting GTP binding . These results reveal an unexpected regulatory pathway that controls R-Ras through an SH3 domain interaction . This pathway appears to be important for the ability of R-Ras to control cell adhesion. J Biol Chem, 2000 Feb 18, 275(7), 5214 - 21 Identification of a novel sterol-independent regulatory element in the human low density lipoprotein receptor promoter; Liu J et al.; The cytokine oncostatin M (OM) activates human low density lipoprotein receptor (LDLR) gene transcription through a sterol-independent mechanism . Previous studies conducted in our laboratory have narrowed the OM-responsive element to promoter region -52 to +13, which contains the repeat 3 and two TATA-like sequences . We now identify LDLR promoter region -17 to -1 as a sterol-independent regulatory element (SIRE) that is critically involved in OM-, transcription factor CCAAT/enhancer-binding protein (C/EBP)-, and second messenger cAMP-mediated activation of LDLR transcription . The SIRE sequence overlaps the previously described TATA-like element and consists of an active C/EBP-binding site (-17 to -9) and a functional cAMP-responsive element (CRE) (-8 to -1) . We demonstrate that (a) mutations within either the C/EBP or CRE site have no impact on basal or cholesterol-mediated repression of LDLR transcription, but they completely abolish OM-mediated activation of LDLR transcription; (b) replacing the repeat 3 sequence that contains the Sp1-binding site with a yeast transcription factor GAL4-binding site in the LDLR promoter construct does not affect OM inducibility, thereby demonstrating that OM induction is mediated through the SIRE sequence in conjunction with a strong activator bound to the repeat 3 sequence; (c) electrophoretic mobility shift and supershift assays confirm the specific binding of transcription factors C/EBP and cAMP-responsive element-binding protein to the SIRE; (d) cotransfection of a human C/EBPbeta expression vector (pEF-NFIL6) with the LDLR promoter construct pLDLR234 increases LDLR promoter activity; and (e) OM and dibutyryl cAMP synergistically activate LDLR transcription through this regulatory element . This study identifies, for the first time, a cis-acting regulatory element in the LDLR promoter that is responsible for sterol-independent regulation of LDLR transcription. J Biol Chem, 2000 Feb 18, 275(7), 4965 - 72 Human peroxisomal multifunctional enzyme type 2 . Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity; Qin YM et al.; Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity . Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532 . To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast . After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized . The purified proteins have similar secondary structural elements, with the same subunit composition . The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive . Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins . The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1. Mol Cell Biol, 2000 Mar, 20(5), 1855 - 67 Mouse Zac1, a transcriptional coactivator and repressor for nuclear receptors; Huang SM et al.; Transcriptional activation by nuclear hormone receptors is mediated by the 160-kDa family of nuclear receptor coactivators . These coactivators associate with DNA-bound nuclear receptors and transmit activating signals to the transcription machinery through two activation domains . In screening for mammalian proteins that bind the C-terminal activation domain of the nuclear receptor coactivator GRIP1, we identified a new variant of mouse Zac1 which we call mZac1b . Zac1 was previously discovered as a putative transcriptional activator involved in regulation of apoptosis and the cell cycle . In yeast two-hybrid assays and in vitro, mZac1b bound to GRIP1, to CREB-binding protein (CBP) and p300 (which are coactivators for nuclear receptors and other transcriptional activators), and to nuclear receptors themselves in a hormone-independent manner . In transient-transfection assays mZac1b exhibited a transcriptional activation activity when fused with the Gal4 DNA binding domain, and it enhanced transcriptional activation by the Gal4 DNA binding domain fused to GRIP1 or CBP fragments . More importantly, mZac1b was a powerful coactivator for the hormone-dependent activity of nuclear receptors, including androgen, estrogen, glucocorticoid, and thyroid hormone receptors . However, with some reporter genes and in some cell lines mZac1b acted as a repressor rather than a coactivator of nuclear receptor activity . Thus, mZac1b can interact with nuclear receptors and their coactivators and play both positive and negative roles in regulating nuclear receptor function. Mol Cell Biol, 2000 Mar, 20(5), 1784 - 96 Sequestration and inhibition of Daxx-mediated transcriptional repression by PML; Li H et al.; PML fuses with retinoic acid receptor alpha (RARalpha) in the t(15;17) translocation that causes acute promyelocytic leukemia (APL) . In addition to localizing diffusely throughout the nucleoplasm, PML mainly resides in discrete nuclear structures known as PML oncogenic domains (PODs), which are disrupted in APL and spinocellular ataxia cells . We isolated the Fas-binding protein Daxx as a PML-interacting protein in a yeast two-hybrid screen . Biochemical and immunofluorescence analyses reveal that Daxx is a nuclear protein that interacts and colocalizes with PML in the PODs . Reporter gene assay shows that Daxx drastically represses basal transcription, likely by recruiting histone deacetylases . PML, but not its oncogenic fusion PML-RARalpha, inhibits the repressor function of Daxx . In addition, SUMO-1 modification of PML is required for sequestration of Daxx to the PODs and for efficient inhibition of Daxx-mediated transcriptional repression . Consistently, Daxx is found at condensed chromatin in cells that lack PML . These data suggest that Daxx is a novel nuclear protein bearing transcriptional repressor activity that may be regulated by interaction with PML. Mol Cell Biol, 2000 Mar, 20(5), 1733 - 46 A combinatorial code for gene expression generated by transcription factor Bach2 and MAZR (MAZ-related factor) through the BTB/POZ domain; Kobayashi A et al.; Bach2 is a B-cell- and neuron-specific transcription repressor that forms heterodimers with the Maf-related oncoproteins . We show here that Bach2 activates transcription by interacting with its novel partner MAZR . MAZR was isolated by the yeast two-hybrid screen using the BTB/POZ domain of Bach2 as bait . Besides the BTB/POZ domain, MAZR possesses Zn finger motifs that are closely related to those of the Myc-associated Zn finger (MAZ) protein . MAZR mRNA was coexpressed with Bach2 in B cells among hematopoietic cells and in developing mouse limb buds, suggesting a cooperative role for MAZR and Bach2 in these cells . MAZR forms homo- and hetero-oligomers with Bach2 through the BTB domain, which oligomers bind to guanine-rich sequences . Unlike MAZ, MAZR functioned as a strong activator of the c-myc promoter in transfection assays with B cells . However, it does not possess a typical activation domain, suggesting a role for it as an unusual type of transactivator . The fgf4 gene, which regulates morphogenesis of limb buds, contains both guanine-rich sequences and a Bach2 binding site in its regulatory region . In transfection assays using fibroblast cells, the fgf4 gene was upregulated in the presence of both MAZR and Bach2 in a BTB/POZ domain-dependent manner . The results provide a new perspective on the function of BTB/POZ domain factors and indicate that BTB/POZ domain-mediated oligomers of transcription factors may serve as combinatorial codes for gene expression. Mol Cell Biol, 2000 Mar, 20(5), 1723 - 32 Overexpression of kinase-associated phosphatase (KAP) in breast and prostate cancer and inhibition of the transformed phenotype by antisense KAP expression; Lee SW et al.; Accumulating evidence suggests that phosphatases play an important role in regulating a variety of signal transduction pathways that have a bearing on cancer . The kinase-associated phosphatase (KAP) is a human dual-specificity protein phosphatase that was identified as a Cdc2- or Cdk2-interacting protein by a yeast two-hybrid screening, yet the biological significance of these interactions remains elusive . We have identified the KAP gene as an overexpressed gene in breast and prostate cancer by using a phosphatase domain-specific differential-display PCR strategy . Here we report that breast and prostate malignancies are associated with high levels of KAP expression . The sublocalization of KAP is variable . In normal cells, KAP is primarily found in the perinuclear region, but in tumor cells, a significant portion of KAP is found in the cytoplasm . Blocking KAP expression by antisense KAP in a tetracycline-regulatable system results in a reduced population of S-phase cells and reduced Cdk2 kinase activity . Furthermore, lowering KAP expression led to inhibition of the transformed phenotype, with reduced anchorage-independent growth and tumorigenic potential in athymic nude mice . These findings suggest that therapeutic intervention might be aimed at repression of KAP gene overexpression in human breast and prostate cancer. Mol Cell Biol, 2000 Mar, 20(5), 1639 - 48 Two novel Drosophila TAF(II)s have homology with human TAF(II)30 and are differentially regulated during development; Georgieva S et al.; TFIID is a multiprotein complex composed of the TATA binding protein (TBP) and TBP-associated factors (TAF(II)s) . The binding of TFIID to the promoter is the first step of RNA polymerase II preinitiation complex assembly on protein-coding genes . Yeast (y) and human (h) TFIID complexes contain 10 to 13 TAF(II)s . Biochemical studies suggested that the Drosophila (d) TFIID complexes contain only eight TAF(II)s, leaving a number of yeast and human TAF(II)s (e.g., hTAF(II)55, hTAF(II)30, and hTAF(II)18) without known Drosophila homologues . We demonstrate that Drosophila has not one but two hTAF(II)30 homologues, dTAF(II)16 and dTAF(II)24, which are encoded by two adjacent genes . These two genes are localized in a head-to-head orientation, and their 5' extremities overlap . We show that these novel dTAF(II)s are expressed and that they are both associated with TBP and other bona fide dTAF(II)s in dTFIID complexes . dTAF(II)24, but not dTAF(II)16, was also found to be associated with the histone acetyltransferase (HAT) dGCN5 . Thus, dTAF(II)16 and dTAF(II)24 are functional homologues of hTAF(II)30, and this is the first demonstration that a TAF(II)-GCN5-HAT complex exists in Drosophila . The two dTAF(II)s are differentially expressed during embryogenesis and can be detected in both nuclei and cytoplasm of the cells . These results together indicate that dTAF(II)16 and dTAF(II)24 may have similar but not identical functions. Mol Cell Biol, 2000 Mar, 20(5), 1616 - 25 Cubitus interruptus requires Drosophila CREB-binding protein to activate wingless expression in the Drosophila embryo; Chen Y et al.; CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways . The Drosophila homologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophila development, including the hedgehog (hh), decapentaplegic (dpp), and Toll pathways . Although dCBP is required for the expression of the hh target genes, wingless (wg) and patched (ptc) in vivo, and potentiates ci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity . We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions . These mutant proteins are unable to transactivate a reporter gene regulated by ci binding sites and have a lower dCBP-stimulated activity than wild-type CI . When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression . Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells . Taken together, our data suggest that dCBP function is necessary for ci-mediated transactivation of wg during Drosophila embryogenesis. Mol Cell Biol, 2000 Mar, 20(5), 1604 - 15 The putative pore-forming domain of Bax regulates mitochondrial localization and interaction with Bcl-X(L); Nouraini S et al.; Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to and uses mitochondria as its major site of action . Bax normally resides in the cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and it promotes apoptosis in two ways: (i) by disrupting mitochondrial membrane barrier function by formation of ion-permeable pores in mitochondrial membranes and (ii) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain and inhibiting their functions . A hairpin pair of amphipathic alpha-helices (alpha5-alpha6) in Bax has been predicted to participate in membrane insertion and pore formation by Bax . We mutagenized several charged residues in the alpha5-alpha6 domain of Bax, changing them to alanine . These substitution mutants of Bax constitutively localized to mitochondria and displayed a gain-of-function phenotype when expressed in mammalian cells . Furthermore, substitution of 8 out of 10 charged residues in the alpha5-alpha6 domain of Bax resulted in a loss of cytotoxicity in yeast but a gain-of-function phenotype in mammalian cells . The enhanced function of this Bax mutant was correlated with increased binding to Bcl-X(L), through a BH3-independent mechanism . These observations reveal new functions for the alpha5-alpha6 hairpin loop of Bax: (i) regulation of mitochondrial targeting and (ii) modulation of binding to antiapoptotic Bcl-2 proteins. Mol Cell Biol, 2000 Mar, 20(5), 1515 - 25 Complex protein interactions within the human polyadenylation machinery identify a novel component; Takagaki Y et al.; Polyadenylation of mRNA precursors is a two-step reaction requiring multiple protein factors . Cleavage stimulation factor (CstF) is a heterotrimer necessary for the first step, endonucleolytic cleavage, and it plays an important role in determining the efficiency of polyadenylation . Although a considerable amount is known about the RNA binding properties of CstF, the protein-protein interactions required for its assembly and function are poorly understood . We therefore first identified regions of the CstF subunits, CstF-77, CstF-64, and CstF-50, required for interaction with each other . Unexpectedly, small regions of two of the subunits participate in multiple interactions . In CstF-77, a proline-rich domain is necessary not only for binding both other subunits but also for self-association, an interaction consistent with genetic studies in Drosophila . In CstF-64, a small region, highly conserved in metazoa, is responsible for interactions with two proteins, CstF-77 and symplekin, a nuclear protein of previously unknown function . Intriguingly, symplekin has significant similarity to a yeast protein, PTA1, that is a component of the yeast polyadenylation machinery . We show that multiple factors, including CstF, cleavage-polyadenylation specificity factor, and symplekin, can be isolated from cells as part of a large complex . These and other data suggest that symplekin may function in assembly of the polyadenylation machinery. J Chromatogr B Biomed Sci Appl, 1999 Dec 10, 735(2), 271 - 7 Comparison of different elution conditions for the immunopurification of recombinant hepatitis B surface antigen; Ibarra N et al.; An immunoaffinity chromatographic method was developed using a mAb immunosorbent to purify recombinant hepatitis B surface antigen (r-HBsAg) from yeast . Elution conditions using a mAb-coated ELISA were improved to select the best conditions to purify r-HBsAg . The optimum results in terms of total quantitative recovery were obtained using 20 mM Tris pH 11.6 . An increase in the CB.Hep-1 mAb (anti-HBsAg) useful immunosorbents half-life and in its yield per cycle was obtained when alkaline elution conditions were used . Moreover, the basic conditions do not affect either the antigenic characteristics or the purity or the molecular integrity of r-HBsAg. Science, 2000 Feb 11, 287(5455), 989 - 94 Conservation and novelty in the evolution of cell adhesion and extracellular matrix genes; Hutter H et al.; New proteins and modules have been invented throughout evolution . Gene "birth dates" in Caenorhabditis elegans range from the origins of cellular life through adaptation to a soil habitat . Possibly half are "metazoan" genes, having arisen sometime between the yeast-metazoan and nematode-chordate separations . These include basement membrane and cell adhesion molecules implicated in tissue organization . By contrast, epithelial surfaces facing the environment have specialized components invented within the nematode lineage . Moreover, interstitial matrices were likely elaborated within the vertebrate lineage . A strategy for concerted evolution of new gene families, as well as conservation of adaptive genes, may underlie the differences between heterochromatin and euchromatin. DNA Seq, 1997, 8(3), 181 - 7 Dissection of the 5.5 Mbp region directly telomeric of HLA-B including a long range restriction map, YAC and PAC contigs; Volz A et al.; A large number of diseases are associated with the human major histocompatibility (HLA) complex located in 6p21.3 . The underlying defect of most of these has not yet been determined even after detailed analysis of the HLA region . Due to the extended haplotypes found in this area, several of the HLA-linked disease genes may be located also telomeric of the class I region . In order to analyse the area covering the 4 megabases directly telomeric of HLA-F in close detail, we have generated 50 new markers . These and other markers have been used to establish a SalI restriction map from 46 YACs . A subset of 42 markers was applied to construct a genomic long range restriction map from an HLA-A2/B13 haplotype . Both maps have been compared revealing the presence of additional 150 kb in the HLA-A2 haplotype close to the RFP locus . Additionally, 47 PACs have been selected mapping to this region and grouped into 7 contigs . Sequencing of these PAC contigs has already been initiated. DNA Seq, 1997, 8(3), 147 - 50 Isolation and characterisation of cosmids to intervals within a 4.5Mb region at 6p21.3; Jazwinska EC et al.; The gene responsible for hereditary haemochromatosis (HH) has recently been identified . One mutation in this gene, termed HFE, has been found in all Australian HH patients . We previously identified a predominant HH ancestral haplotype covering 4.5Mb at 6p21.3, and showed that patients with two copies of this haplotype express a more severe form of the disorder . One key question to now be resolved is why haplotype related variation in phenotypic expression of HH is present if all patients tested have the same HFE mutation . A cosmid resource covering the 4.5Mb HH ancestral haplo type region was obtained . These cosmids provide the material for the completion of a transcript map of this region, and will assist the identification of candidate modifiers of HFE expression. RNA, 2000 Jan, 6(1), 136 - 58 The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates; Bachi A et al.; Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs) . To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family . Mex67p is essential for mRNA export in yeast . Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE) . Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC) . TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes . The nucleoporin-binding domain of TAP comprises residues 508-619 . In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim . Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors . Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs . Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5 . The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC. RNA, 2000 Jan, 6(1), 66 - 78 Interaction cloning and characterization of RoBPI, a novel protein binding to human Ro ribonucleoproteins; Bouffard P et al.; Human Ro ribonucleoproteins (RNPs) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (Ro60) associated with one hY RNA (hY1-5) . Using a modified yeast three-hybrid system, named RNP interaction trap assay (RITA), we cloned a novel Ro RNP-binding protein (RoBPI), based on its property to interact in vivo in yeast with an RNP complex made of recombinant Ro60 (rRo60) protein and hY5 (rhY5) RNA . RoBPI cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end . The 2.0-kb RoBPI mRNA was detected in all human tissues tested . Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice . Two-hybrid, three-hybrid, and RITA experiments respectively established that 60 kDa RoBPI did not interact in yeast with rRo60 alone, with rhY5 RNA alone, or with bait RNPs consisting of rRo60 and recombinant hY1, hY3, or hY4 RNAs . RoBPI coimmunoprecipitated with Ro RNPs from HeLa cell extracts and partially colocalized with Ro60 in nuclei of cultured cells . Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but RoBPI seems more conserved, their interaction may represent a gain of function for Ro RNPs . Alternatively, interaction of RohY5 RNPs with RoBPI may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these RNPs. Ann N Y Acad Sci, 1999, 886, 23 - 36 Trichostatin and leptomycin . Inhibition of histone deacetylation and signal-dependent nuclear export; Yoshida M et al.; Trichostatin A (TSA), an inhibitor of the eukaryotic cell cycle and an inducer of morphological reversion of transformed cells, inhibits histone deacetylase (HDAC) at nanomolar concentrations . Recently, trapoxin, oxamflatin, and FR901228, antitumor agents structurally unrelated to TSA, were found to be potent HDAC inhibitors . These inhibitors activate expression of p21Waf1 and 16INK4A in a p53-independent manner . Changes in the expression of these cell cycle regulators by an increase in histone acetylation may be responsible for cell cycle arrest and antitumor activity by HDAC inhibitors . The target molecule of leptomycin B (LMB), a potent antitumor agent, was genetically and biochemically identified as CRM1, a protein reported as being required for chromosome structure control . We showed that CRM1 was a receptor for the nuclear export signal (NES) and that LMB inhibited nuclear export of proteins . Using LMB, we identified a novel NES in fission yeast transcription factor Pap1, the function of which is abolished by oxidative stress in a manner conserved in eukaryotes. Nucleic Acids Res, 2000 Mar 1, 28(5), 1145 - 53 Regulation of double-strand break-induced mammalian homologous recombination by UBL1, a RAD51-interacting protein; Li W et al.; Mammalian RAD51 protein plays essential roles in DNA homologous recombination, DNA repair and cell proliferation . RAD51 activities are regulated by its associated proteins . It was previously reported that a ubiquitin-like protein, UBL1, associates with RAD51 in the yeast two-hybrid system . One function of UBL1 is to covalently conjugate with target proteins and thus modify their function . In the present study we found that non-conjugated UBL1 forms a complex with RAD51 and RAD52 proteins in human cells . Overexpression of UBL1 down-regulates DNA double-strand break-induced homologous recombination in CHO cells and reduces cellular resistance to ionizing radiation in HT1080 cells . With or without overexpressed UBL1, most homologous recombination products arise by gene conversion . However, overexpression of UBL1 reduces the fraction of bidirectional gene conversion tracts . Overexpression of a mutant UBL1 that is incapable of being conjugated retains the ability to inhibit homologous recombination . These results suggest a regulatory role for UBL1 in homologous recombination. J Virol, 2000 Mar, 74(5), 2073 - 83 The rice tungro bacilliform virus gene II product interacts with the coat protein domain of the viral gene III polyprotein; Herzog E et al.; Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein . The function of most of the viral proteins is still unknown . To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins . P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro . Domains involved in the P2-CP association have been identified and mapped on both proteins . To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants . The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3 . This study suggests that P2 could participate in RTBV capsid assembly. J Virol, 2000 Mar, 74(5), 2067 - 72 Interaction of the cauliflower mosaic virus coat protein with the pregenomic RNA leader; Guerra-Peraza O et al.; Using the yeast three-hybrid system, the interaction of the Cauliflower mosaic virus (CaMV) pregenomic 35S RNA (pgRNA) leader with the viral coat protein, its precursor, and a series of derivatives was studied . The purine-rich domain in the center of the pgRNA leader was found to specifically interact with the coat protein . The zinc finger motif of the coat protein and the preceding basic domain were essential for this interaction . Removal of the N-terminal portion of the basic domain led to loss of specificity but did not affect the strength of the interaction . Mutations of the zinc finger motif abolished not only the interaction with the RNA but also viral infectivity . In the presence of the very acidic C-terminal domain, which is part of the preprotein but is not present in the mature CP, the interaction with the RNA was undetectable. Int J Radiat Biol, 2000 Jan, 76(1), 23 - 9 Production of chromatid breaks by single dsb: evidence supporting the signal model; Rogers-Bald M et al.; PURPOSE: The signal model proposes that all chromatid breaks arise from a single DNA double strand break (dsb) via a recombinational exchange mechanism . Here the prediction that chromatid breaks arise from a single dsb is tested . METHOD: The genetically engineered Chinese hamster cell line GS19-43 containing a unique yeast I-SceI recognition site was treated with I-SceI endonuclease (Meganuclease) in the presence of the porating agent streptolysin O . Chromatid breaks were scored at 4h, chromosome breaks at 18 and 22h following treatment (cells used for a 4h fixation were prelabelled with BrdU over two cell-cycles) . Positive controls were treated with the restriction endonuclease Pst 1 . RESULTS: I-SceI endonuclease produced chromatid breaks and at higher enzyme concentrations isochromatid breaks but no chromatid interchanges . About 16% of the chromatid breaks had a 'colour-switch' between the sister-chromatids at the site of breakage, as revealed by FPG staining . At the longer fixation times (18 and 22 h) chromosome breaks were observed, but again no interchanges were seen . Chromatid and chromosome breaks always appeared on the same chromosome . CONCLUSIONS: The production of chromatid breaks from a single dsb fulfils the prediction of the signal model . Moreover, the production of colour-switch breaks at a similar frequency to that for ionizing radiation indicates that chromatid breaks are produced via recombinational exchanges, a significant proportion of which occurs between sister chromatids . The majority is intrachromatid, not involving strand-switches . The absence of interchromosomal exchanges at all fixation times indicates a requirement of two dsb in two different chromosomes for their formation. Dev Genes Evol, 2000 Feb, 210(2), 105 - 9 A recombinogenic targeting method to modify large-inserts for cis-regulatory analysis in transgenic mice: construction and expression of a 100-kb, zebrafish Hoxa-11b-lacZ reporter gene; Chiu CH et al.; The identification of cis-sequences responsible for spatiotemporal patterns of gene expression often requires the functional analysis of large genomic regions . In this study a 100-kb zebrafish Hoxa-11b-lacZ reporter gene was constructed and expressed in transgenic mice . PAC clone 10-O19, containing a portion of the zebrafish HoxA-b cluster, was captured into the yeast-bacterial shuttle vector, pPAC-ResQ, by recombinogenic targeting . A lacZ reporter gene was then inserted in-frame into exon 1 of the zfHoxa-11b locus by a second round of recombinogenic targeting . Expression of the zfHoxa-11b-lacZ reporter gene in 10.5 d.p.f . transgenic mouse embryos was observed only in the posterior portion of the A-P axis, in the paraxial mesoderm, neural tube, and somites . These findings demonstrate the utility of recombinogenic targeting for the modification and expression of large inserts captured from P1/PAC clones. Plant Cell, 2000 Feb, 12(2), 291 - 300 A new family of high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis; Gillissen B et al.; In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane . Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes . Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins . AtPUP1 transports adenine and cytosine with high affinity . Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport . Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake . Inhibition by cytokinins is competitive (competitive inhibition constant K(i) = 20 to 35 microM), indicating that cytokinins are transported by this system . AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem . The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives. Plant Cell, 2000 Feb, 12(2), 265 - 78 The role of AHA motifs in the activator function of tomato heat stress transcription factors HsfA1 and HsfA2; Doring P et al.; Using reporter assays in tobacco protoplasts and yeast, we investigated the function of the acidic C-terminal activation domains of tomato heat stress transcription factors HsfA1 and HsfA2 . Both transcription factors contain short, essential peptide motifs with a characteristic pattern of aromatic and large hydrophobic amino acid residues embedded in an acidic context (AHA motifs) . The prototype is the AHA1 motif of HsfA2, which has the sequence DDIWEELL . Our mutational analysis supports the important role of the aromatic and large hydrophobic amino acid residues in the core positions of the AHA motifs . The pattern suggests the formation of an amphipathic, negatively charged helix as the putative contact region with components of the basal transcription complex . In support of this concept, proline or positively charged residues in or adjacent to the AHA motifs markedly reduce or abolish their activity . Both AHA motifs of HsfA1 and HsfA2 contribute to activator potential, and they can substitute for each other; however, there is evidence for sequence and positional specificity. Plant Cell, 2000 Feb, 12(2), 249 - 64 Structural domains and matrix attachment regions along colinear chromosomal segments of maize and sorghum; Tikhonov AP et al.; Although a gene's location can greatly influence its expression, genome sequencing has shown that orthologous genes may exist in very different environments in the genomes of closely related species . Four genes in the maize alcohol dehydrogenase (adh1) region represent solitary genes dispersed among large repetitive blocks, whereas the orthologous genes in sorghum are located in a different setting surrounded by low-copy-number DNAs . A specific class of DNA sequences, matrix attachment regions (MARs), was found to be in comparable positions in the two species, often flanking individual genes . If these MARs define structural domains, then the orthologous genes in maize and sorghum should experience similar chromatin environments . In addition, MARs were divided into two groups, based on the competitive affinity of their association with the matrix . The "durable" MARs retained matrix associations at the highest concentrations of competitor DNA . Most of the durable MARs mapped outside genes, defining the borders of putative chromatin loops . The "unstable" MARs lost their association with the matrix under similar competitor conditions and mapped mainly within introns . These results suggest that MARs possess both domain-defining and regulatory roles . Miniature inverted repeat transposable elements (MITEs) often were found on the same fragments as the MARs . Our studies showed that many MITEs can bind to isolated nuclear matrices, suggesting that MITEs may function as MARs in vivo. J Med Genet, 2000 Feb, 37(2), 125 - 7 5p14 deletion associated with microcephaly and seizures; Johnson EI et al.; We report on a father and son who have an interstitial deletion of 5p14 . The father is clinically and mentally normal while the son has significant clinical involvement including microcephaly, seizures, and global developmental delay . The extent of the 5p14 deletion was determined using fluorescence in situ hybridisation (FISH) . The deletion in this present family is smaller than a deletion previously described in a multigenerational family that lacks any clinical phenotype . This report shows that a 5p14 deletion does not always lead to a normal phenotype. Mol Cell Biol Res Commun, 1999 Sep-Dec, 2(3), 150 - 4 PAP I interacts with itself, PAP II, PAP III, and lithostathine/regIalpha; Bodeker H et al.; PAP I, PAP II, PAP III, and lithostathine/regIalpha are members of a multigenic family of proteins expressed in several tissues . PAP I was shown to be antiapoptotic, mitogenic, and anti-inflammatory and can promote cell adhesion to the extracellular matrix . Lithostathine/regIalpha can be mitogenic . Because polymerization might regulate activity, we examined the ability of rat PAP I to interact with itself (homodimerization), PAP II, PAP III, and lithostathine/regIalpha (heterodimerization) by the yeast two-hybrid system, affinity experiments, and crosslinking . PAP I interacted significantly with all members of the PAP protein family, homodimerization showing the strongest interaction as judged by the beta-galactosidase test . This was confirmed by showing specific affinity between a MBP-rPAP I fusion protein and the native rPAP I . Finally, crosslinking experiments showed that rPAP I formed dimers in solution . These findings should be taken into account in functional studies involving PAP I and PAP-related proteins. Genomics, 2000 Jan 1, 63(1), 145 - 8 MAGOH interacts with a novel RNA-binding protein; Zhao XF et al.; MAGOH is the human homologue of Drosophila mago nashi, a protein that is required for normal germ plasm development in the Drosophila embryo . Using human MAGOH as a bait protein in a yeast two-hybrid screen, we recovered four independent cDNA clones that encode different lengths of a novel protein containing a conserved RNA-binding region . This gene, designated RBM8, encodes a 173-aa protein that was shown to have an apparent molecular mass of 26 kDa, as demonstrated by in vitro translation assay . The interaction between MAGOH and RBM8 was demonstrated by both yeast two-hybrid and GST fusion protein pull-down assays . Like MAGOH, RBM8 gene is expressed ubiquitously in human tissues; three species of RBM8 mRNA were detected . Also similar to MAGOH, RBM8 expression is serum inducible in quiescent NIH3T3 fibroblast cells . Genomics, 2000 Jan 1, 63(1), 139 - 41 A new zinc ribbon gene (ZNRD1) is cloned from the human MHC class I region; Fan W et al.; Eleven unique cDNA fragments were identified from YAC B30H3, which spans 330 kb in the human major histocompatibility complex class I region . One fragment (CAT80) was mapped 80 kb telomeric to the HLA-A locus . Using this cDNA fragment as probe, Northern analysis reveals a ubiquitously expressed transcript of about 850 nt in all 16 tissues tested . Based on the cDNA fragment sequence, a full-length cDNA of 858 bp that contains an open reading frame of 378 bp was cloned . Within the putative polypeptide of 126 amino acids, two zinc-ribbon domains were identified: Cx2Cx15Cx2C at the N-terminal and Cx2Cx24Cx2C at the C-terminal . The C-terminal domain is well conserved throughout evolution, including archaea, yeast, Drosophila, nematodes, amphibians, and mammals . The conserved amino acid sequence, CxRCx6Yx3QxRSADEx2TxFxCx2C, is highly homologous to the yeast RNA polymerase A subunit 9 and transcription-associated proteins . Alignment with genomic DNA demonstrates that this gene spans 3.6 kb and consists of four exons and three introns . Cross-species Northern analysis reveals a mouse homolog of a similar size and with an expression profile similar to those of the human gene . We have named this gene ZNRD1 for zinc ribbon domain-containing 1 protein . Genomics, 2000 Jan 1, 63(1), 60 - 8 Physical map of 17p13 and the genes adjacent to p53; Cousin P et al.; Genetic lesions in the p53 tumor suppressor gene are the most frequently observed alterations in human cancers . Typically in tumors, one allele of the p53 gene is initially mutated, followed by deletion of the remaining wildtype allele . In human colon cancer, for example, approximately 70% of late stage tumors are hemizygous mutant p53 . Since the precise gene environment surrounding the p53 gene is not known, the neighboring genes concomitantly lost with wildtype p53 deletion remain undetermined . A restriction enzyme map and clone array of 1.1 Mb surrounding the p53 gene were constructed using a combination of YAC, BAC, NotI linking, and NotI jumping clones . The resulting physical map and clone array include approximately 400 kb telomeric and 700 kb centromeric to the p53 gene . Sequence determination and analysis adjacent to NotI and AscI sites, indicative of CpG islands, allowed the rapid identification of numerous genes within the cloned region . Twenty-seven transcription units were identified, including 18 characterized genes . Limited analysis of primary human colon tumors, hemizygous for the p53 gene, indicates loss of the entire 1.1-Mb region upon deletion of wildtype p53 . Nippon Ishinkin Gakkai Zasshi, 2000, 41(1), 17 - 21 A case of cutaneous Exophiala spinifera infection; Oba M et al.; A 66-year-old female had a solitary dark-red nodule measuring 1 cm in diameter on her left forearm . She often had trauma to her hands and arms . A histopathologic examination demonstrated granulomatous changes in the dermis . Under the high-power magnification yeast-like cells and short toruloid hyphal elements were observed in the granuloma . A few giant cells contained fungal elements . No sclerotic cells were found . On the basis of the histopathologic and mycologic findings, the lesion was diagnosed as a dematiaceous fungal infection caused by Exophiala spinifera . She was treated with oral itraconazole (200 mg/day) and topical heat therapy . The lesion was clinically improved within 58-days . However, E . spinifera was still isolated from the excisional specimen 92-days later . We believe that surgical excision is the choice of therapy if the lesion is small. J Biol Chem, 2000 Feb 11, 275(6), 4467 - 74 PILRalpha, a novel immunoreceptor tyrosine-based inhibitory motif-bearing protein, recruits SHP-1 upon tyrosine phosphorylation and is paired with the truncated counterpart PILRbeta; Mousseau DD et al.; SHP-1-mediated dephosphorylation of protein tyrosine residues is central to the regulation of several cell signaling pathways, the specificity of which is dictated by the intrinsic affinity of SH2 domains for the flanking sequences of phosphotyrosine residues . By using a modified yeast two-hybrid system and SHP-1 as bait, we have cloned a human cDNA, PILRalpha, encoding a 303-amino acid immunoglobulin-like transmembrane receptor bearing two cytoplasmic tyrosines positioned within an immunoreceptor tyrosine-based inhibitory motif . Substrate trapping in combination with pervanadate treatment of 293T cells confirms that PILRalpha associates with SHP-1 in vivo upon tyrosine phosphorylation . Mutation of the tyrosine residues in PILRalpha indicates the pivotal role of the Tyr-269 residue in recruiting SHP-1 . Surface plasmon resonance analysis further suggests that the association between PILRalpha-Tyr-269 and SHP-1 is mediated primarily via the amino-terminal SH2 domain of the latter . Polymerase chain reaction amplification of cDNA in combination with genomic sequence analysis revealed a second gene, PILRbeta, coding for a putative activating receptor as suggested by a truncated cytoplasmic tail and a charged lysine residue in its transmembrane region . The PILRalpha and PILRbeta genes are localized to chromosome 7 which is in contrast with the mapping of known members of the inhibitory receptor superfamily. J Biol Chem, 2000 Feb 11, 275(6), 4383 - 90 Inhibition of nuclear factor-kappaB-mediated transcription by association with the amino-terminal enhancer of split, a Groucho-related protein lacking WD40 repeats; Tetsuka T et al.; The amino-terminal enhancer of split (AES) encodes a 197-amino acid protein that is homologous to the NH(2)-terminal domain of the Drosophila Groucho protein but lacks COOH-terminal WD40 repeats . Although the Drosophila Groucho protein and its mammalian homologs, transducin-like enhancer of split proteins, are known to act as non-DNA binding corepressors, the role of the AES protein remains unclarified . Using the yeast two-hybrid system, we have identified the protein-protein interaction between AES and the p65 (RelA) subunit of the transcription factor nuclear factor kappaB (NF-kappaB), which activates various target genes involved in inflammation, apoptosis, and embryonic development . The interaction between AES and p65 was confirmed by in vitro glutathione S-transferase pull down assay and by in vivo co-immunoprecipitation study . In transient transfection assays, AES repressed p65-driven gene expression . AES also inhibited NF-kappaB-dependent gene expression induced by tumor necrosis factor alpha, interleukin-1beta, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, which is an upstream kinase for NF-kappaB activation . These data indicate that AES acts as a corepressor for NF-kappaB and suggest that AES may play a pivotal role in the regulation of NF-kappaB target genes. J Biol Chem, 2000 Feb 11, 275(6), 4171 - 6 HIV-2 and SIV nef proteins target different Src family SH3 domains than does HIV-1 Nef because of a triple amino acid substitution; Collette Y et al.; The nef gene is required for optimal viral spread of human and simian immunodeficiency viruses . However, the molecular mechanisms underlying the action of the Nef proteins may not be identical for all viral families . Here we investigate the interaction between the Nef protein of human and simian immunodeficiency viruses and SH3 domains from Src family kinases . Using the yeast two-hybrid system and immunoblotting we show that, in contrast to HIV-1 Nef, SIV and HIV-2 Nef poorly interact with Hck SH3 but bind to Src and Fyn SH3 domains . The molecular basis of these differences in SH3 targeting was revealed by sequence analysis and homology modeling of the putative SH3-Nef structures . Three amino acids (Trp-113, Thr-117, and Gln-118) that localize in a "hydrophobic pocket" implicated in SH3 binding of HIV-1 Nef, are systematically substituted in SIV/HIV-2 alleles (by Tyr, Glu, and Glu, respectively) . We demonstrate that site-directed mutagenesis of these residues in SIV(mac239) Nef suffices to restore Hck SH3 binding and co-immunoprecipitation with full-length Hck from transfected cells . Our findings identify fundamental mechanistic differences in targeting of Src family kinases by HIV and SIV Nef . The herein described mechanism of SH3 selection by Nef via a "pocket" proximal to the canonical proline-rich motif may be a common feature for SH3 recognition by their natural ligands. J Biol Chem, 2000 Feb 11, 275(6), 4033 - 40 A functional interaction between dorsal and components of the Smt3 conjugation machinery; Bhaskar V et al.; To identify proteins that regulate the function of Dorsal, a Drosophila Rel family transcription factor, we employed a yeast two-hybrid screen to search for genes encoding Dorsal-interacting proteins . Six genes were identified, including two that encode previously known Dorsal-interacting proteins (Twist and Cactus), three that encode novel proteins, and one that encodes Drosophila Ubc9 (DmUbc9), a protein thought to conjugate the ubiquitin-like polypeptide Smt3 to protein substrates . We have found that DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal . In cultured cells, DmUbc9 was found to relieve inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription . The effect of DmUbc9 on Dorsal activity was potentiated by the overexpression of DmSmt3 . We have also identified a DmSmt3-activating enzyme, DmSAE1/DmSAE2 and found that it further potentiates Dorsal-mediated activation. J Biol Chem, 2000 Feb 11, 275(6), 3867 - 72 Human and mouse Fas (APO-1/CD95) death receptor genes each contain a p53-responsive element that is activated by p53 mutants unable to induce apoptosis; Munsch D et al.; p53 is a tumor suppressor protein that induces apoptosis at least in part through its ability to act as a sequence-specific transactivator . This work reports that intron 1 of the mouse Fas death receptor gene contains a p53-responsive element (p53RE) that matches the p53 consensus sequence and that is located between nucleotides +1704 and +1723 from the transcription initiation site . This element is specifically bound by p53 and functions as a p53-dependent enhancer in mammalian or in yeast reporter gene assays . Contrary to bax, another known pro-apoptotic p53-target gene, both mouse and human FAS p53REs are still activated by the discriminatory p53 mutants Pro-175 and Ala-143, a class of mutants unable to induce apoptosis . We propose that p53-dependent up-regulation of Fas does not induce apoptosis per se but sensitizes the cell to other pro-apoptotic signal(s) . The functional conservation of p53-dependent Fas up-regulation argues strongly in favor of its biological importance and suggests that murine models may be used to study further the in vivo role of Fas in the p53 response. Curr Biol, 2000 Jan 13, 10(1), 55 - 8 The p24 family member p23 is required for early embryonic development; Denzel A et al.; The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation . Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex . Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast . In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature) . In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality . Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members . The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules . The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development . It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway. Curr Biol, 2000 Jan 13, 10(1), 31 - 4 A structurally defined mini-chromosome vector for the mouse germ line; Shen MH et al.; Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors . Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures . Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line . In these experiments, however, the structure and sequence organization of the fragments was not defined . Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression . Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line. Cancer Lett, 1999 Dec 1, 147(1-2), 139 - 47 A new member of the GTPase superfamily that is upregulated in highly metastatic cells; Nakaji T et al.; Two sublines of B16 melanoma cells, F10 and BL6, are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection . We found a new member of the GTPase superfamily, namely TIB929, which displayed an induction of expression in BL6 cells . It conserved three consensus sequences for GTP-binding site motifs and showed a significant homology to the yeast Gtr2 gene throughout the coding sequence . TIB929 was expressed ubiquitously in human tumor cells, with a marked expression in highly metastatic cells . TIB929 was mapped on mouse chromosome 4D, syntenic to human chromosome 1p . The results suggested an involvement of TIB929 in malignant progression. Mol Plant Microbe Interact, 2000 Feb, 13(2), 191 - 202 NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid; Zhou JM et al.; NPR1 is a critical component of the salicylic acid (SA)-mediated signal transduction pathway leading to the induction of defense genes, such as the pathogenesis-related (PR)-1 gene, and enhanced disease resistance . Using a yeast two-hybrid screen, we identified several NPR1-interacting proteins (NIPs) . Two of these NIPs are members of the TGA/OBF family of basic leucine zipper (bZIP) transcription factors; this family has been implicated in the activation of SA-responsive genes, including PR-1 . Six TGA family members were tested and shown to differentially interact with NPR1: TGA2 and TGA3 showed strong affinity for NPR1; TGA5 and TGA6 exhibited weaker affinity; and TGA1 and TGA4 displayed little or no detectable interaction with NPR1, respectively . Interestingly, the amino-termini of these factors were found to decrease their stability in yeast and differentially affect their apparent affinity toward NPR1 . The interacting regions on NPR1 and the TGA factors were also defined . Each of four point mutations in NPR1 that disrupt SA signaling in Arabidopsis completely blocked interaction of NPR1 with TGA2 and TGA3 . TGA2 and TGA3 were also found to bind the SA-responsive element of the Arabidopsis PR-1 promoter . These results directly link NPR1 to SA-induced PR-1 expression through members of the TGA family of transcription factors. Equine Vet J Suppl, 1999 Jul, 30, 605 - 9 The effects of an endurance ride on metabolism and neutrophil function; Jensen-Waern M et al.; The effects of an endurance ride on neutrophil functions in endurance-trained horses were evaluated and related to metabolic changes and changes in cortisol concentrations . Blood samples were taken from 7 horses (aged 9-15 years) one day before, and then 30-60 min, 1 day and 8 days after the ride . The race resulted in elevated serum cortisol levels (< 465 nmol/l) and an increased neutrophil:lymphocyte ratio . Immediately post race, the neutrophil ability to engulf yeast was increased . One day after the race, a decrease in leukotriene B4 production (approximately 40%) and in the respiratory burst (approximately 75%) was observed . Blood glucose concentration remained unchanged, as did serum lactate, which was low . After the race, the muscle glycogen stores were about 33% of the values noted after 8 days of recovery . Histochemical staining showed that 22 +/- 2% of the muscle fibres were totally depleted of glycogen . CK activity increased significantly from 40 +/- 10 mmol/min pre-race to 228 +/- 38 mmol/min post race . It was concluded that both hormonal and metabolic changes occurred during an endurance ride, which not only triggered neutrophil activation, but also induced alterations in their functional capacities. Biosystems, 1999 Dec, 54(1-2), 65 - 70 Dynamic patterns in the locomotion and feeding behaviors by the placozoan Trichoplax adhaerence; Ueda T et al.; The placozoan Trichoplax adhaerence is one of the most primitive multi-cellular organisms, and moves about accompanying perpetual changes in its shape . Changes in position, locomotion velocity and the outer shape of the organism were monitored quantitatively with use of a computer image analysis, and their dynamic patterns in free locomotion and upon feeding were analyzed in terms of non-linear dynamics . The organism changed its behavioral patterns discontinuously in response to various concentrations of yeast extracts (food) . (1) At low concentrations, the organism moved fast with perpetual random changes in shape . Both locomotion velocity and shape changes exhibited 1/f fluctuations . (2) At high concentrations, the shape of the organism as well as the locomotion exhibited oscillations with periods of about 8 min . These limit cycle oscillations bifurcated into the period 2 at the highest concentration tested . The organism flattened more strongly and the locomotion was more reduced on the whole at higher concentrations . (3) At the intermediate concentrations, two patterns as monitored above appeared: one pattern continued for a while and switched to the other abruptly . (4) The average square displacement of the organism increased linearly with time in all cases, indicating that the locomotion is a Brownian movement . In this way, the feeding behaviors by the placozoan are organized as successive co-operative transitions among non-linear dynamic states. FASEB J, 2000 Feb, 14(2), 345 - 54 Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells; Besset S et al.; Sulfation is a major modification of many molecules in eukaryotes that is dependent on the enzymatic synthesis of an activated sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) . While sulfate activation has long been assumed to occur in the cytosol, we show in this study that human PAPS synthetase 1 (PAPSS1), a bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase enzyme sufficient for PAPS synthesis, accumulates in the nucleus of mammalian cells . Nuclear targeting of the enzyme is mediated by its APS kinase domain and requires a catalytically dispensable 21 amino acid sequence at the amino terminus . Human PAPSS1 and Drosophila melanogaster PAPSS localize to the nucleus in yeast and relieve the methionine auxotrophy of ATP sulfurylase- or APS kinase-deficient strains, suggesting that PAPSS1 is fully functional in vivo when targeted to the nucleus . A second PAPS synthetase gene, designated PAPSS2, has recently been described, mutations of which are responsible for abnormal skeletal development in human spondyloepimetaphyseal dysplasia and murine brachymorphism . We found that PAPSS2, which localizes to the cytoplasm when ectopically expressed in mammalian cells, is relocated to the nucleus when coexpressed with PAPSS1 . Taken together, these results indicate that a sulfation pathway might exist in the nucleus of eukaryotic cells . -Besset, S., Vincourt, J.-B., Amalric, F., Girard, J.-P . Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells. FASEB J, 2000 Feb, 14(2), 231 - 41 The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains; Kay BK et al.; Acommon focus among molecular and cellular biologists is the identification of proteins that interact with each other . Yeast two-hybrid, cDNA expression library screening, and coimmunoprecipitation experiments are powerful methods for identifying novel proteins that bind to one's favorite protein for the purpose of learning more regarding its cellular function . These same techniques, coupled with truncation and mutagenesis experiments, have been used to define the region of interaction between pairs of proteins . One conclusion from this work is that many interactions occur over short regions, often less than 10 amino acids in length within one protein . For example, mapping studies and 3-dimensional analyses of antigen-antibody interactions have revealed that epitopes are typically 4-7 residues long (1) . Other examples include protein-interaction modules, such as Src homology (SH) 2 and 3 domains, phosphotyrosine binding domains (PTB), postsynaptic density/disc-large/ZO1 (PDZ) domains, WW domains, Eps15 homology (EH) domains, and 14-3-3 proteins that typically recognize linear regions of 3-9 amino acids . Each of these domains has been the subject of recent reviews published elsewhere (2 3 4 5 6 7) . Among the primary structures of many ligands for protein-protein interactions, the amino acid proline is critical . In particular, SH3, WW, and several new protein-interaction domains prefer ligand sequences that are proline-rich . In addition, even though ligands for EH domains and 14-3-3 domains are not proline-rich, they do include a single proline residue . This review highlights the analysis of those protein-protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.-Kay, B . K., Williamson, M . P., Sudol, M . The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. Biochem J, 2000 Feb 15, 346 Pt 1, 155 - 61 Subcellular localization of proteasomes and their regulatory complexes in mammalian cells; Brooks P et al.; Proteasomes can exist in several different molecular forms in mammalian cells . The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure . Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis . The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g . LMP7) . In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies . Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver . LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations . 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines . Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes . S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm . The REG was found to be localized predominantly in the cytoplasm . Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution . These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes. Nat Biotechnol, 2000 Feb, 18(2), 208 - 12 Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes; Jung W et al.; Isoflavones have drawn much attention because of their benefits to human health . These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation . Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway . To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins . We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet . We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant . Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes. Dev Biol, 2000 Feb 15, 218(2), 314 - 25 Membrane fusion proteins are required for oskar mRNA localization in the Drosophila egg chamber; Ruden DM et al.; We used a genetic screen in Drosophila to identify mutations which disrupt the localization of oskar mRNA during oogenesis . Based on the hypothesis that some cytoskeletal components which are required during the mitotic divisions will also be required for oskar mRNA localization during oogenesis, we designed the following genetic screen . We screened for P-element insertions in genes which slow down the blastoderm mitotic divisions . A secondary genetic screen was to generate female germ-line clones of these potential cell division cycle genes and to identify those which cause the mislocalization of oskar mRNA . We identified mutations in ter94 which disrupt the localization of oskar mRNA to the posterior pole of the oocyte . Ter94 is a member of the CDC48p/VCP subfamily of AAA proteins which are involved in homotypic fusion of the endoplasmic reticulum during mitosis . Consistent with the function of the yeast ortholog, ter94-mutant egg chambers are defective in the assembly of the endoplasmic reticulum . We tested whether other membrane biosynthesis genes are required for localizing oskar mRNA during oogenesis . We found that ovaries that are mutant for syntaxin-1a, rop, and synaptotagmin are also defective in oskar mRNA localization during oogenesis . We suggest a pathway for the role of membrane assembly proteins on oskar mRNA localization . Mol Plant Microbe Interact, 2000 Jan, 13(1), 118 - 24 Molecular cloning and characterization of a tobacco MAP kinase kinase that interacts with SIPK; Liu Y et al.; A tobacco MAP kinase termed SIPK (Salicylic acid-Induced Protein Kinase) is activated in response to a variety of stress signals, including pathogen attack and wounding (S . Zhang and D.F . Klessig, Proc . Natl . Acad . Sci . USA 95:7225-7230, 1998; S . Zhang and D.F . Klessig, Proc . Natl . Acad . Sci . USA 95:7433-7438, 1998) . Using the yeast two-hybrid system, we have identified a gene encoding a protein that interacts with SIPK but not the wounding induced protein kinase (WIPK), which is another tobacco MAP kinase . Sequence analysis indicated that this SIPK-interacting protein is a member of the MAP kinase kinase family; thus, it was named SIPK kinase (SIPKK) . Co-immunoprecipitation experiments demonstrated that SIPKK and SIPK interact in vitro . Consistent with its putative function as a kinase, SIPKK phosphorylated myelin basic protein in vitro . Interestingly, SIPKK was induced at the mRNA level after Tobacco mosaic virus (TMV) infection or wounding, albeit with kinetics that are too slow to account for the activation of SIPK following these stimuli. Mutat Res, 1999 Dec 16, 431(1), 93 - 103 5-methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation; Monti P et al.; Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC) . However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system . In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase . Methylation induced 98% protection to HpaII endonuclease . Unmethylated and methylated pLS76 vectors were then UVC irradiated (lambda(max): 254 nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter . The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P = 0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay . Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation . We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solarlight and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a "spontaneous" origin. Kansenshogaku Zasshi, 1999 Dec, 73(12), 1227 - 31 {A case of acquired immunodeficiency syndrome (AIDS) with histoplasmosis}; Aso Y et al.; Fungal infection is a major opportunistic infection in AIDS . Histoplasmosis is often seen in American AIDS, but only one case has been reported in Japan . We report a AIDS case of with histoplasmosis in Japan . The patient was a forty year old male living in the U.S from 1987 to 1990 . He was diagnosed as candidial esophagitis in July, 1994, and human immunodeficiency virus type 1 (HIV) antibody positive led to a diagnosis of AIDS . He was admitted to our hospital with fever and lymphadenopathy (neck, abdomen) in August . The therapy for candidial esophagitis was successful and he was recovering, but he was newly diagnosed as atypical mycobacteriosis and Kaposi's sarcoma . Though the fever was slight, it persisted . He was discharged from our hospital in October . He was readmitted for a high fever and dehydration in December, but died after a week from disseminated intravascular coagulation (DIC) . Histoplasma capsulatum was found by blood and ascites cultures on second admission . Many yeast like histoplasma cells in granuloma of the liver were found at autopsy . For moderate or severe histoplasmosis, amphotericin B is generally used as the first induction therapy . Fluconazole (FLCZ) is used as a maintenance therapy . We did not use amphotericin B, but used FLCZ because we did not diagnose histoplasmosis before death, and his general condition became worse . The effect of FLCZ therapy was unclear in our case because he had other infections . We expect that AIDS with histoplasimosis will increase in Japan through HIV infected patients infected in the U.S.A. Hum Mol Genet, 2000 Feb 12, 9(3), 395 - 401 Differentially regulated and evolved genes in the fully sequenced Xq/Yq pseudoautosomal region; Ciccodicola A et al.; Human sex chromosomes, which are morphologically and genetically different, share few regions of homology . Among them, only pseudoautosomal regions (PARs) pair and recombine during meiosis . To better address the complex biology of these regions, we sequenced the telomeric 400 kb of the long arm of the human X chromosome, including 330 kb of the human Xq/YqPAR and the telomere . Sequencing reveals subregions with distinctive regulatory and evolutionary features . The proximal 295 kb contains two genes inactivated on both the inactive X and Y chromosomes { SYBL1 and a novel homologue ( HSPRY3 ) of Drosophila sprouty } . The GC-rich distal 35 kb, added in stages and much later in evolution, contains the X/Y expressed gene IL9R and a novel gene, CXYorf1, only 5 kb from the Xq telomere . These properties make Xq/YqPAR a model for studies of region-specific gene inactivation, telomere evolution, and involvement in sex-limited conditions. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1166 - 71 Mice reconstituted with DNA polymerase beta-deficient fetal liver cells are able to mount a T cell-dependent immune response and mutate their Ig genes normally; Esposito G et al.; The ubiquitously expressed, error-prone DNA polymerase beta (polbeta) plays a role in base excision repair, and the involvement of this molecule in the nonhomologous end joining (NHEJ) process of DNA repair has recently been demonstrated in yeast . Polbeta-deficient mice are not viable, and studies on conditional mutants revealed a competitive disadvantage of polbeta(-/-) vs . wild-type cells . We show here that polbeta-deficient mice survive up to day 18.5 postcoitum, but die perinatally; a circumstance that allowed the investigation of a potential role of polbeta in lymphocyte development by transfer of fetal liver cells (FLC) derived from polbeta(-/-) embryos into lethally irradiated hosts . FLC transfers using mutant cells lead to an almost normal reconstitution of the lymphocyte compartment, indicating that polbeta-deficiency does not prevent V(D)J recombination, which is known to employ factors of the NHEJ pathway . Mice reconstituted with polbeta(-/-) FLC mount a normal T cell-dependent immune response against the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP) . Moreover, germinal center B cells from NP-immunized reconstituted mice show normal levels and patterns of somatic point mutations in their rearranged antibody genes, demonstrating that polbeta is not critically involved in somatic hypermutation. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1154 - 9 TAF250 is required for multiple developmental events in Drosophila; Wassarman DA et al.; The TFIID transcription initiation complex is composed of TBP and multiple TAFs . Studies in unicellular systems indicate that TAF250 is required for progression through G(1)/S of the cell cycle and repression of apoptosis . Here we extend these in vivo studies by determining the developmental requirements for TAF250 in a multicellular organism, Drosophila . TAF250 mutants were isolated in a genetic screen that also yielded TAF60 and TAF110 mutants, indicating that TAFs function coordinately to regulate transcription . Null alleles of TAF250 are recessive larval lethal . However, combinations of weak loss-of-function TAF250 alleles survive to adulthood and reveal requirements for TAF250 during ovary, eye, ocelli, wing, bristle, and terminalia development as well as overall growth of the fly . These phenotypes suggest roles for TAF250 in regulating the cell cycle, cell differentiation, cell proliferation, and cell survival . Finally, molecular analysis of TAF250 mutants reveals that the observed phenotypes are caused by mutations in a central region of TAF250 that is conserved among metazoan organisms . This region is contained within the TAF250 histone acetyltransferase domain, but the mutations do not alter the histone acetyltransferase activity of TAF250 in vitro, indicating that some other aspect of TAF250 function is affected . Because this region is not conserved in the yeast TAF250 homologue, TAF145, it may define an activity for TAF250 that is unique to higher eukaryotes. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1125 - 30 The sentrin-conjugating enzyme mUbc9 interacts with GLUT4 and GLUT1 glucose transporters and regulates transporter levels in skeletal muscle cells; Giorgino F et al.; Glucose transport in insulin-regulated tissues is mediated by the GLUT4 and GLUT1 transporters . Using the yeast two-hybrid system, we have cloned the sentrin-conjugating enzyme mUbc9 as a protein that interacts with the GLUT4 COOH-terminal intracellular domain . The mUbc9 enzyme was found to bind directly to GLUT4 and GLUT1 through an 11-aa sequence common to the two transporters and to modify both transporters covalently by conjugation with the mUbc9 substrate, sentrin . Overexpression of mUbc9 in L6 skeletal muscle cells decreased GLUT1 transporter abundance 65%, resulting in decreased basal glucose transport . By contrast, mUbc9 overexpression increased GLUT4 abundance 8-fold, leading to enhanced transport stimulation by insulin . A dominant-negative mUbc9 mutant lacking catalytic activity had effects opposite to those of wild-type mUbc9 . The regulation of GLUT4 and GLUT1 was specific, as evidenced by an absence of mUbc9 interaction with or regulation of the GLUT3 transporter isoform in L6 skeletal muscle cells . The mUbc9 sentrin-conjugating enzyme represents a novel regulator of GLUT1 and GLUT4 protein levels with potential importance as a determinant of basal and insulin-stimulated glucose uptake in normal and pathophysiological states. Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 1056 - 61 Identification of a transcriptional repressor related to the noncatalytic domain of histone deacetylases 4 and 5; Zhou X et al.; Histone deacetylases (HDACs) are involved in regulating transcription by modifying the core histones of the nucleosome . To date, six HDACs have been identified in mammalian cells: the yeast RPD3 homologs HDAC1, 2, and 3 and the yeast HDA1 homologs HDAC4, 5, and 6 . HDAC4 and HDAC5 contain a noncatalytic N-terminal domain . Herein, we report the identification of a protein HDRP (HDAC-related protein) that shares 50% identity in deduced amino acid sequence to the noncatalytic N-terminal domain of HDAC4 and 5 . The steady-state levels of HDRP mRNA are high in human brain, heart, and skeletal muscle and low in the several other tissues . HDRP has an apparent molecular mass of approximately 75 kDa . HDRP does not possess intrinsic HDAC activity but forms complexes with both HDAC1 and HDAC3 . HDRP represses both basal and activated transcription in transient transfection assays when tethered to DNA as a Gal4-fusion protein . HDAC inhibitors do not reverse transcriptional repression mediated by Gal4-HDRP . Thus, HDRP is a transcriptional repressor and can repress transcription in the presence of HDAC inhibitors. Genetics, 2000 Feb, 154(2), 787 - 801 A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2alpha; Sood R et al.; A family of protein kinases regulates translation in response to different cellular stresses by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha) . In yeast, an eIF-2alpha kinase, GCN2, functions in translational control in response to amino acid starvation . It is thought that uncharged tRNA that accumulates during amino acid limitation binds to sequences in GCN2 homologous to histidyl-tRNA synthetase (HisRS) enzymes, leading to enhanced kinase catalytic activity . Given that starvation for amino acids also stimulates phosphorylation of eIF-2alpha in mammalian cells, we searched for and identified a GCN2 homologue in mice . We cloned three different cDNAs encoding mouse GCN2 isoforms, derived from a single gene, that vary in their amino-terminal sequences . Like their yeast counterpart, the mouse GCN2 isoforms contain HisRS-related sequences juxtaposed to the kinase catalytic domain . While GCN2 mRNA was found in all mouse tissues examined, the isoforms appear to be differentially expressed . Mouse GCN2 expressed in yeast was found to inhibit growth by hyperphosphorylation of eIF-2alpha, requiring both the kinase catalytic domain and the HisRS-related sequences . Additionally, lysates prepared from yeast expressing mGCN2 were found to phosphorylate recombinant eIF-2alpha substrate . Mouse GCN2 activity in both the in vivo and in vitro assays required the presence of serine-51, the known regulatory phosphorylation site in eIF-2alpha . Together, our studies identify a new mammalian eIF-2alpha kinase, GCN2, that can mediate translational control. Genetics, 2000 Feb, 154(2), 713 - 24 Functional domains of the Drosophila bicaudal-D protein; Oh J et al.; The localization of oocyte-specific determinants in the form of mRNAs to the pro-oocyte is essential for the establishment of oocyte identity . Localization of the Bicaudal-D (Bic-D) protein to the presumptive oocyte is required for the accumulation of Bic-D and other mRNAs to the pro-oocyte . The Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins, and several of the mutations in Bic-D map to these conserved domains . We have undertaken a structure-function analysis of Bic-D by testing the function of mutant Bic-D transgenes (Bic-D(H)) deleted for each of the heptad repeat domains in a Bic-D null background . Our transgenic studies indicate that only the C-terminal heptad repeat deletion results in a protein that has lost zygotic and ovarian functions . The three other deletions result in proteins with full zygotic function, but with affected ovarian function . The functional importance of each domain is well correlated with its conservation in evolution . The analysis of females heterozygous for Bic-D(H) and the existing alleles Bic-D(PA66) or Bic-D(R26) reveals that Bic-D(R26) as well as some of Bic-D(H) transgenes have antimorphic effects . The yeast two-hybrid interaction assay shows that Bic-D forms homodimers . Furthermore, we found that Bic-D exists as a multimeric protein complex consisting of Egl and at least two Bic-D monomers. Genetics, 2000 Feb, 154(2), 657 - 68 Mutational analysis of a histone deacetylase in Drosophila melanogaster: missense mutations suppress gene silencing associated with position effect variegation; Mottus R et al.; For many years it has been noted that there is a correlation between acetylation of histones and an increase in transcriptional activity . One prediction, based on this correlation, is that hypomorphic or null mutations in histone deacetylase genes should lead to increased levels of histone acetylation and result in increased levels of transcription . It was therefore surprising when it was reported, in both yeast and fruit flies, that mutations that reduced or eliminated a histone deacetylase resulted in transcriptional silencing of genes subject to telomeric and heterochromatic position effect variegation (PEV) . Here we report the first mutational analysis of a histone deacetylase in a multicellular eukaryote by examining six new mutations in HDAC1 of Drosophila melanogaster . We observed a suite of phenotypes accompanying the mutations consistent with the notion that HDAC1 acts as a global transcriptional regulator . However, in contrast to recent findings, here we report that specific missense mutations in the structural gene of HDAC1 suppress the silencing of genes subject to PEV . We propose that the missense mutations reported here are acting as antimorphic mutations that "poison" the deacetylase complex and propose a model that accounts for the various phenotypes associated with lesions in the deacetylase locus. Oncogene, 2000 Jan 20, 19(3), 351 - 7 Self-association of the SET domains of human ALL-1 and of Drosophila TRITHORAX and ASH1 proteins; Rozovskaia T et al.; The human ALL-1 gene is involved in acute leukemia through gene fusions, partial tandem duplications or a specific deletion . Several sequence motifs within the ALL-1 protein, such as the SET domain, PHD fingers and the region with homology to DNA methyl transferase are shared with other proteins involved in transcription regulation through chromatin alterations . However, the function of these motifs is still not clear . Studying ALL-1 presents an additional challenge because the gene is the human homologue of Drosophila trithorax . The latter is a member of the trithorax-Polycomb gene family which acts to determine the body pattern of Drosophila by maintaining expression or repression of the Antennapedia-bithorax homeotic gene complex . Here we apply yeast two hybrid methodology, in vivo immunoprecipitation and in vitro 'pull down' techniques to show self association of the SET motifs of ALL-1, TRITHORAX and ASH1 proteins (Drosophila ASH1 is encoded by a trithorax-group gene) . Point mutations in evolutionary conserved residues of TRITHORAX SET, abolish the interaction . SET-SET interactions might act in integrating the activity of ALL-1 (TRX and ASH1) protein molecules, simultaneously positioned at different maintenance elements and directing expression of the same or different target genes. Eur J Nutr, 1999 Oct, 38(5), 241 - 6 Investigations on the intestinal availability of native thiamin in selected foods and feedstuffs; Roth-Maier DA et al.; The aim of the present study was to investigate the precaecal digestibility as a quantitative measure for the intestinal availability of naturally occurring thiamin from selected foods and feedstuffs . Therefore, three experiments were conducted to examine the following foods and feedstuffs: Eggs, bananas, white cabbage, corn, milk, fish, barley, soybeans, rice, wheat bran, brewer's yeast, rye and soybean meal . The foods and food processing procedures were made with regard to their relevance in human and animal nutrition . For all experiments male pigs with an initial live weight between 33 and 40 kg were fitted with an end-to-end ileo-rectal anastomosis with preserved ileo-caeco-colicvalve . Three weeks after surgery, the digestibility trials were carried out from week 4 to week 9 and week 12 to week 17 after surgery . The animals were fed the individual experimental diets for a period of 12 days while digesta were collected twice a day quantitatively during the final 5 days of this period . Precaecal digestibility for thiamin from all tested foods and feedstuffs was within a range from 73% to 94% with the highest values from boiled soybeans, boiled rice and barley, and the lowest value from steamed fish . In comparison with the animal products the plant products show on average a nearly equal precaecal digestibility for thiamin (87.3% versus 83.5%) . Moreover, all tested foods and feedstuffs exhibit a relatively good intestinal availability of thiamin. J Bioenerg Biomembr, 1999 Oct, 31(5), 419 - 30 Uncoupling protein--a useful energy dissipator; Klingenberg M; The structure/function relationship in the uncoupling proteins (UCP) is reviewed, stressing UCP from brown adipose tissue (UCP1) since, so far, nearly no biochemistry is known for the UCP variants UCP2, UCP3, and UCP4 . The transport for H+ and Cl- and its dependence on fatty acids in reconstituted vesicles is described . The inhibition and binding of nucleotides to UCP1, in particular, the pH dependence and two-stage binding are analyzed . A model for the role of fatty acid in H+ transport is shown . The role of specific residues in UCP1 is analyzed by directed mutagenesis in a yeast expression system . The different regulation by the cellular energy potential of UCP1 versus UCP3 is discussed. J Bioenerg Biomembr, 1999 Oct, 31(5), 407 - 18 Contribution to the identification and analysis of the mitochondrial uncoupling proteins; Ricquier D et al.; This review is primarily focused on the contribution of our laboratory to study of the mitochondrial uncoupling UCPs . The initial stage was the description of a 32-kDa membranous protein specifically induced in brown adipose tissue mitochondria of cold-adapted rats . This protein was then shown by others to be responsible for brown fat thermogenesis and was referred to as the uncoupling protein-UCP (recently renamed UCP1) . cDNA and genomic clones of UCP1 were isolated and used to investigate the topology and functional organization of the protein in the membrane and the mechanisms of control of UCP1 gene transcription . Orientation of the transmembrane fragments was proposed and specific amino acid residues involved in the inhibition of UCP1 by purine nucleotides were identified in recombinant yeast . A potent enhancer mediating the response of the UCP1 gene to retinoids and controlling the specific transcription in brown adipocytes was identified using transgenic mice . More recently, we identified UCP2, an UCP homolog widely expressed in human and rodent tissues we also collaborated to characterize the plant UCP . Although the biochemical activities and physiological roles of the novel UCPs are not well understood, these recent data stimulate research on mitochondrial carriers, mitochondrial bioenergetics, and energy expenditure. J Biol Chem, 2000 Feb 4, 275(5), 3355 - 9 Differential regulation of sentrinized proteins by a novel sentrin-specific protease; Gong L et al.; Sentrin-1, also called SUMO-1, is a protein of 101 residues that is distantly related to ubiquitin and another ubiquitin-like protein, NEDD8 . Here we report the cloning of a novel sentrin-specific protease, SENP1, which has no homology to the known de-ubiquitinating enzymes or ubiquitin C-terminal hydrolases . However, SENP1 is distantly related to the yeast Smt3-specific protease, Ulp1 . A COS cell expression system was used to demonstrate the activity of SENP1 in vivo . When HA-tagged sentrin-1 was co-expressed with SENP1, the higher molecular weight sentrin-1 conjugates were completely removed . Surprisingly, the major sentrinized band at 90 kDa remained intact . The disappearance of the high molecular weight sentrin-1 conjugates also coincided with an increase in free sentrin-1 monomers . SENP1 is also active against proteins modified by sentrin-2, but not those modified by ubiquitin or NEDD8 . In addition, sentrinized PML, a tumor suppressor protein that resides in the nucleus, was selectively affected by SENP1, whereas sentrinized RanGAP1, which is associated with the cytoplasmic fibrils of the nuclear pore complex, remained intact . The inability of SENP1 to process sentrinized RanGAP1 in vivo is most likely due to its nuclear localization because SENP1 is active against sentrinized RanGAP1 in vitro . The identification of a nuclear-localized, sentrin-specific protease will provide a unique tool to study the role of sentrinization in the biological function of PML and in the pathogenesis of acute promyelocytic leukemia. J Biol Chem, 2000 Feb 4, 275(5), 3305 - 12 The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties; Felts SJ et al.; The hsp90 family of molecular chaperones was expanded recently due to the cloning of TRAP1 and hsp75 by yeast two-hybrid screens . Careful analysis of the human TRAP1 and hsp75 sequences revealed that they are identical, and we have cloned a similar protein from Drosophila . Immunofluorescence data show that human TRAP1 is localized to mitochondria . This mitochondrial localization is supported by the existence of mitochondrial localization sequences in the amino termini of both the human and Drosophila proteins . Due to the striking homology of TRAP1 to hsp90, we tested the ability of TRAP1 to function as an hsp90-like chaperone . TRAP1 did not form stable complexes with the classic hsp90 co-chaperones p23 and Hop (p60) . Consistent with these observations, TRAP1 had no effect on the hsp90-dependent reconstitution of hormone binding to the progesterone receptor in vitro, nor could it substitute for hsp90 to promote maturation of the receptor to its hormone-binding state . However, TRAP1 is sufficiently conserved with hsp90 such that it bound ATP, and this binding was sensitive to the hsp90 inhibitor geldanamycin . In addition, TRAP1 exhibited ATPase activity that was inhibited by both geldanamycin and radicicol . Thus, TRAP1 has functions that are distinct from those of hsp90. J Biol Chem, 2000 Feb 4, 275(5), 3150 - 7 Biochemical analysis of the arginine methylation of high molecular weight fibroblast growth factor-2; Klein S et al.; The post-translational methylation of the N-terminally extended or high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) has been shown to affect the nuclear accumulation of the growth factor . In this study, we determined the extent and position of methyl groups in HMW FGF-2 . Using mass spectrometry and amino acid sequence analysis, we have shown that the 22- and 22.5-kDa forms of HMW FGF-2 contain five dimethylated arginines located at positions -22, -24, -2