Exp Cell Res, 2000 May 25, 257(1), 22 - 32 Cloning and characterization of DIP1, a novel protein that is related to the Id family of proteins; Yao Y et al.; Using human cyclin D1 as the "bait" in a yeast two-hybrid system, together with a HL60 cDNA library, we identified a novel human nuclear protein designated DIP1 . This protein is expressed in a variety of cell types, and in fibroblasts its level remains constant throughout the cell cycle . However, the level of this protein increases severalfold during the differentiation of HL60 cells . The DIP1 protein can be phosphorylated in vitro by a cellular kinase and this activity reaches its maximum in extracts obtained from cells in the G1 phase of the cell cycle . DIP1 contains a helix-loop-helix motif but lacks an adjacent basic DNA-binding domain, thus resembling the Id family of proteins . The dip1 gene is located on human chromosome 16p11.2-12, a locus that is amplified in several types of human cancer . These results suggest that DIP1 may be involved in the control of gene expression and differentiation, but its precise function remains to be determined. Curr Genet, 2000 May, 37(5), 328 - 32 Mutation of a putative AMPK phosphorylation site abolishes the repressor activity but not the nuclear targeting of the fungal glucose regulator CRE1; Vautard-Mey G et al.; In filamentous ascomycetes, glucose repression is mediated by CRE1, a zinc-finger protein related to Miglp from yeast . Five putative AMPK phosphorylation motifs identified in the glucose repressor from the phytopathogenic fungus Sclerotinia sclerotiorum were mutated in a GFP::CRE1 translational fusion . Complementation experiments in Aspergillus nidulans and fluorescence microscopy analyses showed that mutation of one site (Ser266) abolishes the repressor activity of the fusion protein but not its nuclear targeting, suggesting that an AMPK protein kinase may be involved in the function of the fungal glucose repressor. Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7178 - 83 A role of transcriptional activators as antirepressors for the autoinhibitory activity of TATA box binding of transcription factor IID; Kotani T et al.; The TATA box-binding activity of transcription factor IID (TFIID) is autoinhibited by the N-terminal domain of the Drosophila TATA box-binding protein- (TBP) associated factor 230/yeast TBP-associated factor 145 subunit, which binds to the TATA box-binding domain of TBP by mimicking the TATA box structure . Here, we propose a mechanism of transcriptional activation that involves antirepression of this autoinhibitory activity by transcriptional activators . Like the autoinhibitory domain of TFIID, various acidic activators interact with the TATA box-binding domain of TBP . Moreover, the autoinhibitory domain of TFIID, which is known to interact with only the TATA box-binding domain of TBP, acts as an activation domain when fused to the GAL4 DNA-binding domain, indicating that interaction with the TATA-binding domain of TBP is crucial for activation of transcription . In a reciprocal fashion, the acidic activation domains can function as the autoinhibitory domain when the latter is replaced by the former within TFIID . These results indicate that activation domains and the autoinhibitory domain of TFIID are interchangeable, supporting a role for transcriptional activators as antirepressors of the autoinhibitory activity of the TATA box binding of TFIID. Int J Oncol, 2000 Jul, 17(1), 189 - 95 Binding of the growth suppressor p53 protein to the cell cycle regulator phosphatase cdc25C; Rief N et al.; Human p53 is a growth suppressor which not only functions in mammalian cells but also in fission yeast . It was previously shown that the cell cycle regulating phosphatase cdc25C suppresses the p53 induced growth arrest in fission yeast . In the present study we analysed the mechanism of this suppression . We found that cdc25C directly interacts with p53 . By using different deletion mutants the binding region was narrowed down on the polypeptide chain of p53 to amino acids 287-340 . To test the functional significance we analysed the effect of this interaction on the DNA binding activity of p53 . As shown by band shift experiments binding of cdc25C to p53 does not modify the DNA binding activity of p53 . Our data suggest that the observed suppression of the p53 induced growth arrest by cdc25C might be achieved by direct binding of cdc25C to the C-terminus of p53. J Biol Chem, 2000 Aug 25, 275(34), 26316 - 21 A novel human rad54 homologue, Rad54B, associates with Rad51; Tanaka K et al.; Members of the SNF2/SWI2 family, characterized with sequence motifs similar to those found in DNA and RNA helicases, play roles in various aspects of cellular fundamental processes such as transcriptional regulation, chromosome stability, nucleotide excision repair, and recombination . We have isolated a novel member of the human SNF2/SWI2 family, RAD54B, which is highly homologous to mammalian RAD54 . The RAD54 gene is a member of the RAD52 epistasis group which is involved in the recombinational repair of DNA damage . Here we demonstrate that human Rad54B (hRad54B), like human Rad54 (hRad54), associates with human Rad51 (hRad51) . Both hRad54B and hRad54 associate with hRad51 through their NH(2)-terminal domains, but there are differences in their ways of association with hRad51 . In contrast to Rad54, whose association with Rad51 is induced by ionizing radiation, Rad54B associates with Rad51 constitutively in immunoprecipitation experiments . Also, the failure to detect the interaction between hRad54B and hRad51 in the yeast two-hybrid assay suggests that their interaction, unlike that between hRad54 and hRad51, may be indirect . Immunofluorescence microscopy revealed that hRad54B formed nuclear foci that colocalized with hRad51, hRad54, and BRCA1 . These findings suggest that Rad54B may be functionally distinct from Rad54, although it may play an active role in recombination processes in concert with other members of the RAD52 epistasis group. J Biol Chem, 2000 Aug 25, 275(34), 26158 - 63 Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells; Trepakova ES et al.; Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC) . Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC . Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells . In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets . The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes . Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches . These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion. Blood, 2000 Jun 15, 95(12), 3945 - 50 Differential expression of a novel C-terminally truncated splice form of SMAD5 in hematopoietic stem cells and leukemia; Jiang Y et al.; SMADs are evolutionarily conserved transducers of the differentiation and growth arrest signals from the transforming growth factor/BMP (TGF/BMP) family of ligands . Upon receptor activation, the ligand-restricted SMADs(1-35) are phosphorylated in the C-terminal MH2 domain and recruit the common subunit SMAD4/DPC-4 gene to the nucleus to mediate target gene expression . Frequent inactivating mutations of SMAD4, or less common somatic mutations of SMAD2 seen in solid tumors, suggest that these genes have a suppressor function . However, there have been no identified mutations of SMAD5, although the gene localizes to the critical region of loss in chromosome 5q31.1 (chromosome 5, long arm, region 3, band 1, subband 1) in myelodysplasia (MDS) and acute myelogenous leukemia (AML) . A ubiquitously expressed novel isoform, SMAD5beta, encodes a 351 amino acid protein with a truncated MH2 domain and a unique C-terminal tail of 18 amino acids, which may be the functional equivalent of inactivating mutations . The levels of SMAD5beta transcripts are higher in the undifferentiated CD34(+) hematopoietic stem cells than in the terminally differentiated peripheral blood leukocytes, thereby implicating the beta form in stem cell homeostasis . Yeast 2-hybrid interaction assays reveal the lack of physical interactions between SMAD5beta and SMAD5 or SMAD4 . The expression of SMAD5beta may represent a novel mechanism to protect pluripotent stem cells and malignant cells from the growth inhibitory and differentiation signals of BMPs . (Blood . 2000;95:3945-3950) J Neurosci, 2000 Jun 15, 20(12), 4615 - 26 Lens injury stimulates axon regeneration in the mature rat optic nerve; Leon S et al.; In mature mammals, retinal ganglion cells (RGCs) are unable to regenerate their axons after optic nerve injury, and they soon undergo apoptotic cell death . However, a small puncture wound to the lens enhances RGC survival and enables these cells to regenerate their axons into the normally inhibitory environment of the optic nerve . Even when the optic nerve is intact, lens injury stimulates macrophage infiltration into the eye, Muller cell activation, and increased GAP-43 expression in ganglion cells across the entire retina . In contrast, axotomy, either alone or combined with intraocular injections that do not infringe on the lens, causes only a minimal change in GAP-43 expression in RGCs and a minimal activation of the other cell types . Combining nerve injury with lens puncture leads to an eightfold increase in RGC survival and a 100-fold increase in the number of axons regenerating beyond the crush site . Macrophage activation appears to play a key role, because intraocular injections of Zymosan, a yeast cell wall preparation, stimulated monocytes in the absence of lens injury and induced RGCs to regenerate their axons into the distal optic nerve. J Neurosci, 2000 Jun 15, 20(12), 4596 - 605 Functional interactions between Drosophila bHLH/PAS, Sox, and POU transcription factors regulate CNS midline expression of the slit gene; Ma Y et al.; During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells . CNS midline development is dependent on Single-minded (Sim), a basic-helix-loop-helix (bHLH)-PAS transcription factor . We show here that Fish-hook (Fish), a Sox HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene expression . single-minded, fish-hook, and drifter are all expressed in developing midline cells, and both loss- and gain-of-function assays revealed genetic interactions between these genes . The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells . Fish-hook directly associates with the PAS domain of Single-minded and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast . In addition, Fish can form homodimers and also associates with other bHLH-PAS and POU proteins . These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription factors . Functional interactions between members of these protein families may be important for numerous developmental and physiological processes. J Biol Chem, 2000 Aug 25, 275(34), 26458 - 66 Molecular characterization of human acetyl-CoA synthetase, an enzyme regulated by sterol regulatory element-binding proteins; Luong A et al.; Through suppressive subtractive hybridization, we identified a new gene whose transcription is induced by sterol regulatory element-binding proteins (SREBPs) . The gene encodes acetyl-CoA synthetase (ACS), the cytosolic enzyme that activates acetate so that it can be used for lipid synthesis or for energy generation . ACS genes were isolated previously from yeast, but not from animal cells . Recombinant human ACS was produced by expressing the cloned cDNA transiently in human cells . After purification by nickel chromatography, the 701-amino acid cytosolic enzyme was shown to function as a monomer . The recombinant enzyme produced acetyl-CoA from acetate in a reaction that required ATP . As expected for a gene controlled by SREBPs, ACS mRNA was induced when cultured cells were deprived of sterols and repressed by sterol addition . The pattern of regulation resembled the regulation of enzymes of fatty acid synthesis . ACS mRNA was also elevated in livers of transgenic mice that express dominant-positive versions of all three isoforms of SREBP . We conclude that ACS mRNA, and hence the ability of cells to activate acetate, is regulated by SREBPs in parallel with fatty acid synthesis in animal cells. Genomics, 2000 May 15, 66(1), 110 - 2 Physical map of the region surrounding the OTOFERLIN locus on chromosome 2p22-p23; Yasunaga S et al.; The autosomal recessive form of nonsyndromic deafness DFNB9 has been mapped to a 2-cM region on chromosome 2p22-p23, and the responsible gene, OTOF, has been recently identified by positional cloning combined with a candidate gene approach . In the course of this gene cloning, we established a contig of yeast artificial chromosomes, bacterial artificial chromosomes, and P1 phage artificial chromosomes delimited by polymorphic markers D2S2170 and D2S170, i.e . , extending over approximately 3500 kb . Sixty expressed sequence tags or genes and 14 sequence-tagged sites, 11 of which are polymorphic, were mapped to this contig and assigned to 21 chromosomal intervals . Genomics, 2000 May 15, 66(1), 104 - 9 Large-insert clone/STS contigs in Xq11-q12, spanning deletions in patients with androgen insensitivity and mental retardation; Schueler MG et al.; An integrated large-insert clone map of the region Xq11-q12 is presented . A physical map containing markers within a few hundred kilobases of the centromeric locus DXZ1 to DXS1125 spans nearly 5 Mb in two contigs separated by a gap estimated to be approximately 100-250 kb . The contigs combine 75 yeast artificial chromosome clones, 12 bacterial artificial chromosome clones, and 17 P1-derived artificial chromosome clones with 81 STS or EST markers . Overall marker density across this region is approximately 1 STS/60 kb . Mapped within the contigs are 12 ESTs as well as 5 known genes, moesin (MSN), hephaestin (HEPH), androgen receptor (AR), oligophrenin-1 (OPHN1), and Eph ligand-2 (EPLG2) . Orientation of the contigs on the X chromosome, as well as marker order within the contigs, was unambiguously determined by reference to a number of X chromosome breakpoints . In addition, the distal contig spans deletions from chromosomes of three patients exhibiting either complete androgen insensitivity (CAI) or a contiguous gene syndrome that includes CAI, impaired vision, and mental retardation . Genomics, 2000 May 15, 66(1), 48 - 54 Identification and characterization of YME1L1, a novel paraplegin-related gene; Coppola M et al.; A gene responsible for an autosomal recessive form of hereditary spastic paraplegia (SPG7) was recently identified . This gene encodes paraplegin, a mitochondrial protein highly homologous to the yeast mitochondrial AAA proteases Afg3p, Rca1p, and Yme1p, which have both proteolytic and chaperone-like activities at the inner mitochondrial membrane . By screening the expressed sequence tag database, we identified and characterized a novel human gene, YME1L1 (YME1L1-like1, HGMW-approved symbol) . This gene encodes a predicted protein of 716 amino acids highly similar to all mitochondrial AAA proteases and in particular to yeast Yme1p . Expression and immunofluorescence studies revealed that YME1L1 and paraplegin share a similar expression pattern and the same subcellular localization in the mitochondrial compartment . YME1L1 may represent a candidate gene for other forms of hereditary spastic paraplegia and possibly for other neurodegenerative disorders . Genomics, 2000 May 15, 66(1), 15 - 25 Chromosome translocations in breast cancer with breakpoints at 8p12; Courtay-Cahen C et al.; Unbalanced chromosome translocations with breakpoints around 8p12, resulting in loss of distal 8p, are common in carcinomas . We have mapped the 8p12 breakpoints in three breast cancer cell lines, T-47D, MDA-MB-361, and ZR-75-1, using YACs and PACs between D8S540 and D8S255 by fluorescence in situ hybridization . All three lines had a breakpoint close to D8S505, proximal to HGL . Each breakpoint was distinct, but all were within 0.5 to 1.5 Mb of each other . The T-47D cell line had a straightforward translocation, but in MDA-MB-361 and ZR-75-1 the translocations were accompanied by local rearrangements of surprising complexity . Small regions of 8p from close to the breakpoint were duplicated or amplified as inserts in the attached chromosome fragment . ZR-75-1 also had retained a separate fragment of about 1 Mb, from the region 1 to 3 Mb telomeric to the common breakpoint, that included HGL . This line also had an interstitial deletion several megabases more centromeric . The data suggest that breakpoints on 8p12 are clustered in a small region and show that translocations breaking there may be accompanied by additional rearrangements . Oncogene, 2000 Jun 1, 19(24), 2812 - 9 Degradation of human Aurora2 protein kinase by the anaphase-promoting complex-ubiquitin-proteasome pathway; Honda K et al.; Human Aurora2 was originally identified by its close homology to yeast IPL1 and fly aurora, which are key regulators of chromosome segregation and a family of serine/threonine kinases . Here we demonstrate that the Aurora2 protein is degraded rapidly after G2/M phase release in mammalian cells . Aurora2 protein has a rapid turnover rate with a half-life of approximately 2 h . In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of unstable proteins . The treatment of mammalian cells with proteasome inhibitors blocks Aurora2 degradation . Furthermore, Aurora2 is polyubiquitinated in vivo and in vitro using anaphase-promoting complex (APC) . These results demonstrate that Aurora2 protein is turned over through the APC-ubiquitin-proteasome pathway . Oncogene (2000) 19, 2812 - 2819 Cell, 2000 May 26, 101(5), 471 - 83 Identification of human Rap1: implications for telomere evolution; Li B et al.; It has been puzzling that mammalian telomeric proteins, including TRF1, TRF2, tankyrase, and TIN2 have no recognized orthologs in budding yeast . Here, we describe a human protein, hRap1, that is an ortholog of the yeast telomeric protein, scRap1p . hRap1 has three conserved sequence motifs in common with scRap1, is located at telomeres, and affects telomere length . However, while scRap1 binds telomeric DNA directly, hRap1 is recruited to telomeres by TRF2 . Extending the comparison of telomeric proteins to fission yeast, we identify S . pombe Taz1 as a TRF ortholog, indicating that TRFs are conserved at eukaryotic telomeres . The data suggest that ancestral telomeres, like those of vertebrates, contained a TRF-like protein as well as Rap1 . We propose that budding yeast preserved Rap1 at telomeres but lost the TRF component, possibly concomitant with a change in the telomeric repeat sequence. Cancer Res, 2000 Jun 1, 60(11), 2786 - 9 Translocation t(10;14)(q11.2:q22.1) fusing the kinetin to the RET gene creates a novel rearranged form (PTC8) of the RET proto-oncogene in radiation-induced childhood papillary thyroid carcinoma; Salassidis K et al.; Evaluation of 20 cases of radiation-induced childhood papillary thyroid carcinoma using fluorescence in situ hybridization demonstrated the presence of clonal translocations affecting the RET locus . Semiquantitative reverse transcription-PCR indicated overexpression of the RET tyrosine kinase (TK) domain in four cases . In two cases, the RET rearrangements PTC6 and PTC7 were identified and assigned to balanced translocations t(7;10)(q32;q11.2) and t(1;10)(p13;q11.2), respectively . In one case with a balanced translocation t(10;14)(q11.2;q22.1), 5' rapid amplification of cDNA ends revealed a novel type of RET oncogenic activation (PTC8), arising from a fusion of the 5' part of the kinectin (KTN1) gene to the TK domain of the RET gene . The presence of coiled-coil domains in the resulting ktn1/ret fusion protein suggests ligand-independent dimerization and thus constitutive activation of the ret TK domain. Cancer Res, 2000 Jun 1, 60(11), 2775 - 9 Deletion of 6q16-q21 in human lymphoid malignancies: a mapping and deletion analysis; Jackson A et al.; Two distinct regions of minimal deletion (RMD) have been identified at 6q25-q27 in non-Hodgkin's lymphoma (RMD-1), and at 6q21-q23 in acute lymphoblastic leukemia (ALL; RMD-2) by loss of heterozygosity and fluorescence in situ hybridization studies . In this study, 30 overlapping yeast artificial chromosomes (YACs), 1 expressed sequence tag, and 11 novel YAC ends were identified using bidirectional YAC walks between markers D6S447 (proximal) and D6S246 (distal) in RMD-2 . The genes AF6q21, human homologue of the Drosophila tailless (HTLX), CD24 antigen, the Kruppel-like zinc finger BLIMP1, and cyclin C (CCNC), previously mapped to 6q21, were accurately positioned in a telomere-to-centromere orientation . Approximately 3.5 Mb were found to separate the BLIMP1 (adjacent to D6S447) and AF6q21 genes (telomeric to D6S246) . Deletions of 6q were investigated in 21 cases of ALL using the newly characterized YAC clones in dual-color fluorescence in situ hybridization studies . A region centromeric to D6S447 (containing marker D6S283) and a region telomeric to marker CHLC.GGAT16CO2 (and containing marker D6S268) were identified as distinct and nonoverlapping regions of deletion in ALL. Ther Drug Monit, 2000 Jun, 22(3), 237 - 44 Catalytic activity of three variants (Ile, Leu, and Thr) at amino acid residue 359 in human CYP2C9 gene and simultaneous detection using single-strand conformation polymorphism analysis; Ieiri I et al.; This study evaluated the catalytic activity of three variants (Ile, Leu, and Thr) at codon 359 of CYP2C9 enzymes expressed in a yeast cDNA expression system, and then established single-strand conformation polymorphism (PCR-SSCP) analysis for simultaneous detection as a screening method . Diclofenac was used for the in vitro experiment, and its hydroxy metabolite (4'-hydroxydiclofenac) was measured by HPLC . To discuss the in vivo effect of the Thr359 variant on the pharmacokinetics of phenytoin, a case report is presented . The efficiency of the SSCP method was evaluated by analyzing DNA samples from a homozygote for Ile359 and a heterozygote for Leu359 or Thr359 . To evaluate the interaction between the P450 level and reductase activity, two batches of the Thr359 variant with a different P450:reductase activity ratio (1:4.0 and 1:1.4) were used . The in vitro study revealed that recombinant Ile359, Leu359, and Thr359 (2 batches) possessed a mean Km of 2.0, 16.5 and (3.8 and 2.9) micromol and Vmax of 12.4, 17.9 and (4.4 and 5.1) nmol/min/nmol P450, respectively . Although the magnitude of the change in catalytic efficiency for the Thr359 variant was close to that of the Leu359 variant, the effect of the two variants on diclofenac 4'-hydroxylation appears to be different because Leu359 variant was associated with a high Km, and Thr359 with a low Vmax . No significant differences in the kinetic data were observed between the two Thr359 enzymes, suggesting that low reductase activity in the Thr359 enzyme was not a major determinant in the present in vitro experiment . Estimated pharmacokinetic parameters of phenytoin obtained by the Bayesian method in an epileptic patient who was a heterozygote carrier for Thr359 variant were: Km = 6.45 microg/mL, Vmax = 5.77 mg/kg/d, and Vmax/Km = 0.89 L/kg/day . The Vmax/Km value in this patient was similar to the population mean value (0.90 L/kg/day) in Japanese heterozygotes for the Leu359 variant . Results for PCR-SSCP were in complete agreement with those obtained using established methods . Thus, the PCR-SSCP approach is useful for identifying these three variants of the CYP2C9 gene. Appl Biochem Biotechnol, 2000 Spring, 84-86, 1147 - 61 Brazilian bioethanol program; Zanin GM et al.; Brazil is the largest producer of bioethanol, and sugarcane is the main raw material . Bioethanol is produced by both batch and continuous processes, and in some cases, flocculating yeast is used . This article analyzes the Brazilian Ethanol Program . For the 1996-1997 harvest, Brazil produced 14.16 billion L of ethanol and 13.8 million metric t of sugar, from 286 million metric t of sugarcane . These products were produced by 328 industries in activity, with 101 autonomous ethanol plants producing only ethanol, and 227 sugar mills producing sugar and ethanol . The sugar-ethanol market reaches about 7.5 billion US$/yr, accounting for direct and indirect revenues. Int J Androl, 2000, 23 Suppl 2, 92 - 4 Temporal control of protein synthesis during spermatogenesis; Braun RE; During oogenesis and spermatogenesis transcription ceases prior to the differentiation of the mature cells . To complete germ cell differentiation and initiate early embryogenesis, proteins are synthesized from pre-existing mRNAs that are stored for several days . It is well established that important regulatory elements functioning in spatial localization, temporal translation or messenger RNA stability are located in the 3' untranslated region (3' UTR) of mRNAs . During mammalian spermatogenesis temporal translational regulation of the protamine 1 (Prm1) mRNA is dependent on a highly conserved sequence located in the distal region of its 3' UTR . The 17-nucleotide translational control element (TCE) mediates translational repression of the Prm1 mRNA . Mutation of the TCE causes premature synthesis of protamine protein and sterility . The Prm1 mRNA is stored as a cytoplasmic ribonucleoprotein (mRNP) particle in spermatids . Contained within the particle are several members of the Y box family of nucleic acid binding proteins . In the yeast three-hybrid system the murine Y box proteins MSY1, MSY2 and MSY4 bind in a sequence-dependent manner to a conserved region in the proximal portion of the Prm1 3' UTR . Sequence-specific binding by MSY4 to the Y box recognition sequence (YRS) is dependent on the highly conserved cold shock domain, possibly through the RNP1 and RNP2 motifs present within it . The Y box proteins may function as translational repressors in vivo . Alternatively, their primary function may be to protect mRNAs from degradation during their extended period of storage . Translational activation of stored mRNAs is essential for the completion of gametogenesis . Proper translational activation of the Prm1 mRNA in elongated spermatids requires the cytoplasmic double-stranded RNA binding protein TARBP2 . Tarbp2 is expressed at low levels in many cells but is expressed at robust levels in late stage meiotic cells and in postmeiotic spermatids . Mice mutant for Tarbp2 are defective in proper translational activation of the Prm1 and Prm2 mRNAs and are sterile . Current studies are designed to determine the mechanism by which proteins bound to the 3' UTR communicate with the 5' end of the message to control translational silencing and activation. Plant J, 2000 May, 22(4), 355 - 65 Isolation and characterization of HsfA3, a new heat stress transcription factor of Lycopersicon peruvianum; Bharti K et al.; Stress-induced transcription of heat shock proteins (Hsps) in eukaryotes is mediated by a conserved class of transcription factors called heat stress transcription factors (Hsfs) . Here we report the isolation and functional characterization of HsfA3, a new member of the Hsf family . HsfA3 was cloned from a tomato heat stress cDNA library by yeast two-hybrid screening, using HsfA1 as a bait . HsfA3 is a single-copy gene with all the conserved sequence elements characteristic of a heat stress transcription factor . The constitutively expressed HsfA3 is mainly found in the cytoplasm under control conditions and in the nucleus under heat stress conditions . Functionally, HsfA3 behaves similarly to the already known members of tomato Hsf family . It is able to substitute yeast Hsf for viability functions and is a strong activator of Hsf-dependent reporter constructs both in tobacco protoplasts and yeast . Finally, similar to the AHA motifs in HsfA1 and HsfA2, the activator function depends on four short peptide motifs with a central tryptophan residue found in the C-terminal domain of HsfA3. Eur J Biochem, 2000 Jun, 267(12), 3891 - 901 The PKC targeting protein RACK1 interacts with the Epstein-Barr virus activator protein BZLF1; Baumann M et al.; Phorbol esters reactivate Epstein-Barr virus (EBV) from latently infected cells via transcriptional activation of the viral immediate-early gene BZLF1 . BZLF1 is a member of the extended AP-1 family of transcription factors that binds to specific BZLF1-binding motifs within early EBV promoters and to consensus AP-1 sites . Regulation of BZLF1's activity is achieved at the transcriptional level as well as through post-translational modifications . Recently, we reported that the transcriptional activity of BZLF1 is augmented by TPA {Baumann, M., Mischak, H., Dammeier, S., Kolch, W., Gires, O., Pich, D., Zeidler, R., Delecluse, H . J . & Hammerschmidt, W., (1998) J . Virol . 72, 8105-8114} . The increase of BZLF1's activity depends on a single serine residue (S186) that is phosphorylated by protein kinase C (PKC) in vitro and in vivo after stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) . Here, we identified RACK1 as a binding partner of BZLF1 in a yeast interaction trap assay . RACK stands for receptor of activated C-kinase and is involved in targeting activated PKCs and other signaling proteins . In vivo, RACK1 binds directly to the transactivation domain of BZLF1 . Although a functional relationship between BZLF1 and PKC could be mediated by RACKs, RACK1 did not have a detectable effect on the phosphorylation status of BZLF1 in in vitro or in vivo phosphorylation assays . We suggest that RACK1 may act as a scaffolding protein on BZLF1 independently of activated PKCs. Eur J Biochem, 2000 Jun, 267(12), 3453 - 60 Molecular cloning and functional expression of triterpene synthases from pea (Pisum sativum) new alpha-amyrin-producing enzyme is a multifunctional triterpene synthase; Morita M et al.; Ursane type triterpene is one of the most widespread triterpene aglycones found in plants, together with oleanane type, and these two types often occur together in the same plant . Pisum sativum is known to produce both types of triterpenes . Homology based PCRs with degenerate primers designed from the conserved sequences found in the known beta-amyrin synthases have resulted in cloning of two triterpene synthase cDNAs from immature seeds of P . sativum . They show high sequence identities to each other (78%) and also to the known beta-amyrin synthases (70-90%) . ORFs of the full-length clones named as PSY (2277 bp, codes for 759 amino acids) and PSM (2295 bp, codes for 765 amino acids) were ligated into the yeast expression vector pYES2 under the control of GAL1 promoter . Heterologous expression in yeast revealed PSY to be a P . sativum beta-amyrin synthase . Surprisingly, however, PSM turned out to be a novel mixed amyrin synthase producing both alpha- and beta-amyrin . Several minor triterpenes were also identified as the PSM byproducts . The presence of such multifunctional triterpene synthase would account for the co-occurence of ursane and oleanane type triterpenes in plants. Mol Biol Cell, 2000 Jun, 11(6), 2069 - 83 Histone deacetylase inhibitors trigger a G2 checkpoint in normal cells that is defective in tumor cells; Qiu L et al.; Important aspects of cell cycle regulation are the checkpoints, which respond to a variety of cellular stresses to inhibit cell cycle progression and act as protective mechanisms to ensure genomic integrity . An increasing number of tumor suppressors are being demonstrated to have roles in checkpoint mechanisms, implying that checkpoint dysfunction is likely to be a common feature of cancers . Here we report that histone deacetylase inhibitors, in particular azelaic bishydroxamic acid, triggers a G2 phase cell cycle checkpoint response in normal human cells, and this checkpoint is defective in a range of tumor cell lines . Loss of this G2 checkpoint results in the tumor cells undergoing an aberrant mitosis resulting in fractured multinuclei and micronuclei and eventually cell death . This histone deacetylase inhibitor-sensitive checkpoint appears to be distinct from G2/M checkpoints activated by genotoxins and microtubule poisons and may be the human homologue of a yeast G2 checkpoint, which responds to aberrant histone acetylation states . Azelaic bishydroxamic acid may represent a new class of anticancer drugs with selective toxicity based on its ability to target a dysfunctional checkpoint mechanism in tumor cells. Mol Biol Cell, 2000 Jun, 11(6), 2007 - 18 Interaction of the tau2 transcriptional activation domain of glucocorticoid receptor with a novel steroid receptor coactivator, Hic-5, which localizes to both focal adhesions and the nuclear matrix; Yang L et al.; Hic-5 (hydrogen peroxide-inducible clone-5) is a focal adhesion protein that is involved in cellular senescence . In the present study, a yeast two-hybrid screen identified Hic-5 as a protein that interacts with a region of the glucocorticoid receptor that includes a nuclear matrix-targeting signal and the tau2 transcriptional activation domain . In transiently transfected mammalian cells, overexpression of Hic-5 potentiated the activation of reporter genes by all steroid receptors, excluding the estrogen receptor . The activity of the estrogen receptor and the thyroid hormone receptor was stimulated by Hic-5 in the presence but not in the absence of coexpressed coactivator GRIP1 . In biochemical fractionations and indirect immunofluorescence assays, a fraction of endogenous Hic-5 in REF-52 cells and transiently expressed Hic-5 in Cos-1 cells was associated with the nuclear matrix . The C-terminal region of Hic-5, which contains seven zinc fingers arranged in four LIM domains, was required for interaction with focal adhesions, the nuclear matrix, steroid receptors, and the tau2 domain of glucocorticoid receptor . The N-terminal region of Hic-5 possesses a transcriptional activation domain and was essential for the coactivator activity of Hic-5 . Given the coexisting cytoplasmic and nuclear distributions of Hic-5 and its role in steroid receptor-mediated transcriptional activation, it is proposed that Hic-5 might transmit signals that emanate at cell attachment sites and regulate transcription factors, such as steroid receptors. Cell, 2000 Apr 28, 101(3), 271 - 81 act up controls actin polymerization to alter cell shape and restrict Hedgehog signaling in the Drosophila eye disc; Benlali A et al.; Cells in the morphogenetic furrow of the Drosophila eye disc undergo a striking shape change immediately prior to their neuronal differentiation . We have isolated mutations in a novel gene, act up (acu), that is required for this shape change . acu encodes a homolog of yeast cyclase-associated protein, which sequesters monomeric actin; we show that acu is required to prevent actin filament polymerization in the eye disc . In contrast, profilin promotes actin filament polymerization, acting epistatically to acu . However, both acu and profilin are required to prevent premature Hedgehog-induced photoreceptor differentiation ahead of the morphogenetic furrow . These findings suggest that dynamic changes in actin filaments alter cell shape to control the movement of signals that coordinate a wave of differentiation. Mol Endocrinol, 2000 Jun, 14(6), 915 - 25 Activating protein-1, nuclear factor-kappaB, and serum response factor as novel target molecules of the cancer-amplified transcription coactivator ASC-2; Lee SK et al.; ASC-2 was recently discovered as a cancer-amplified transcription coactivator molecule of nuclear receptors, which interacts with multifunctional transcription integrators steroid receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP)/p300 . Herein, we report the identification of three mitogenic transcription factors as novel target molecules of ASC-2 . First, the C-terminal transactivation domain of serum response factor (SRF) was identified among a series of ASC-2-interacting proteins from the yeast two-hybrid screening . Second, ASC-2 specifically interacted with the activating protein-1 (AP-1) components c-Jun and c-Fos as well as the nuclear factor-kappaB (NFkappaB) components p50 and p65, as demonstrated by the glutathione S-transferase pull-down assays as well as the yeast two-hybrid tests . In cotransfection of mammalian cells, ASC-2 potentiated transactivations by SRF, AP-1, and NFkappaB in a dose-dependent manner, either alone or in conjunction with SRC-1 and p300 . In addition, ASC-2 efficiently relieved the previously described transrepression between nuclear receptors and either AP-1 or NFkappaB . Overall, these results suggest that the nuclear receptor coactivator ASC-2 also mediates transactivations by SRF, AP-1, and NFkappaB, which may contribute to the putative, ASC-2-mediated tumorigenesis. Mol Microbiol, 2000 Jun, 36(5), 1148 - 55 Interacting interfaces of the P4 antirepressor E and the P2 immunity repressor C; Eriksson SK et al.; Antirepressors have been identified as proteins interacting with transcriptional repressors leading to expression of the repressed genes . The defective satellite phage/plasmid P4 has the capacity to derepress the unrelated prophage P2 after infection, thereby getting access to the late functions of the helper that are required for P4 lytic growth . The derepression of prophage P2 is mediated by the P4 E protein that function as an antirepressor by binding to the P2 immunity repressor C . A P2 mutant, sos, has been isolated that is insensitive to the action of the P4 E protein . In the present study, we show that sos is a point mutation in the P2 immunity repressor gene C and that it makes P4 E unable to turn the transcriptional switch of P2 from the lysogenic state to the lytic mode in a two plasmid reporter system . Furthermore, the interaction between C and E, when analysed in the yeast two-hybrid system, is blocked by the sos mutation . An analysis of C mutants indicates that the dimerization function of C is located in the C-terminal part of the protein and the dimerization defective mutants are unable to bind to their operator DNA . The sos mutation does not affect the capacity of the protein to dimerize . Using the yeast two-hybrid system, compensatory E mutants have been isolated that can interact with Sos, but they are unable to turn the transcriptional switch controlled by the Sos repressor . However, one point mutation in the E protein is shown to be unable to turn the transcriptional switch controlled by the wild-type C repressor. Kidney Int, 2000 Jun, 57(6), 2221 - 8 Identification of novel genes expressed during metanephric induction through single-cell library screening; Abidari JM et al.; BACKGROUND: Development of the mature kidney is dependent on a series of inductive events between a portion of the epithelial bud at the distal end of the nephric duct and a neighboring domain of committed metanephric mesenchyme . Several genes have been identified to date that are critical in the inductive process . For example, the deletion of Bmp7 from the mouse genome results in dysgenesis or agenesis of the kidney . These findings suggest that Bmp7 controls the expression of genes important for nephrogenesis, but the identity of these genes has remained largely undetermined . METHODS: Single cells were isolated from mouse metanephric mesenchyme during the time of induction (between E11.0 and E11.5) and cDNA libraries constructed from induced and uninduced tissue . Subtractive hybridization was performed to isolate genes that were expressed during E11.5 but not E11.0 . RESULTS: Using this approach, we identified eight previously known genes, three of which were known to be involved in metanephric induction, thus validating our approach, and nine novel genes . Eight of these genes were completely novel, whereas one was similar to a member of the yeast Anaphase Promoting Complex . CONCLUSIONS: Through subtractive hybridization of mouse E11.0 and E11.5 metanephric mesenchyme single-cell cDNA libraries, we have identified novel genes that are candidates for involvement in nephrogenesis through their up-regulation during the inductive process. J Immunol, 2000 Jun 15, 164(12), 6287 - 95 Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function; Junn E et al.; As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen . Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding . mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm . Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX . When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced . TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1 . Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress . To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells . When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells . In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated . Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity. J Immunol, 2000 Jun 15, 164(12), 6188 - 92 Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation; Ling X et al.; Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents . A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein . A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry . Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo . Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro . These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules. Dev Dyn, 2000 Jun, 218(2), 300 - 15 Tubedown-1, a novel acetyltransferase associated with blood vessel development; Gendron RL et al.; We have used an embryonic endothelial cell line (IEM cells) as an experimental system for identifying and characterizing new molecules which are regulated during blood vessel development . A novel gene isolated from IEM cells, tubedown-1 (tbdn-1), is expressed at high levels in unstimulated IEM cells and is downregulated during formation of capillary tube structures by the IEM cells induced by basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) in vitro . Tbdn-1 is also downregulated in M1 myeloid leukemia cells after differentiation in response to LIF in vitro . Tbdn-1 is homologous to the yeast NAT-1 N-terminal acetyltransferases and encodes a novel protein of approximately 69 kDa associated with an acetyltransferase activity . Levels and distribution of tbdn-1 expression are regulated in both endothelial and hematopoietic cells during development in tissues such as the yolk sac blood islands, heart, and liver blood vessels . In the adult, tbdn-1 expression is low or undetected in most organs examined with the exception of the atrial endocardium, the endothelial and myeloid compartments of bone marrow, and the remodeling vascular bed of atretic ovarian follicles . The distribution and regulation of expression of tbdn-1 suggest that this novel acetyltransferase may be involved in regulating vascular and hematopoietic development and physiologic angiogenesis . Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6334 - 9 Analysis of mutations and suppressors affecting interactions between the subunits of the HIV type 1 reverse transcriptase; Tachedjian G et al.; HIV-1 reverse transcriptase (RT) catalyzes the conversion of genomic RNA into cDNA . The enzyme is a heterodimer of p66 and p51 subunits, and the dimerization of these subunits is required for optimal enzyme activity . To analyze this process at the genetic level, we developed constructs that permit the detection of the interaction between these subunits in the yeast two-hybrid system . Genetic analysis of RT subdomains required for heterodimerization revealed that the fingers and palm of p66 were dispensable for p51 interaction . However, as little as a 26-amino acid deletion at the C terminus of p51 prevented dimerization with p66 . A primer grip mutation, L234A, previously shown to inhibit RT dimerization by biochemical assays, also prevented RT dimerization in the yeast two-hybrid system . Second-site mutations that restored RT dimerization in yeast to the L234A parent were recovered in the tryptophan repeat region at the dimer interface and at the polymerase active site, suggesting the involvement of these sites in RT dimerization . In vitro binding experiments confirmed the effects of the L234A mutation and the suppressor mutations on the interaction of the two subunits . The RT two-hybrid assay should facilitate the extensive genetic analysis of RT dimerization and should make possible the rapid screening of potential inhibitors of this essential process. Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6316 - 21 Zinc-bundle structure of the essential RNA polymerase subunit RPB10 from Methanobacterium thermoautotrophicum; Mackereth CD et al.; The RNA polymerase subunit RPB10 displays a high level of conservation across archaea and eukarya and is required for cell viability in yeast . Structure determination of this RNA polymerase subunit from Methanobacterium thermoautotrophicum reveals a topology, which we term a zinc-bundle, consisting of three alpha-helices stabilized by a zinc ion . The metal ion is bound within an atypical CX(2)CX(n)CC sequence motif and serves to bridge an N-terminal loop with helix 3 . This represents an example of two adjacent zinc-binding Cys residues within an alpha-helix conformation . Conserved surface features of RPB10 include discrete regions of neutral, acidic, and basic residues, the latter being located around the zinc-binding site . One or more of these regions may contribute to the role of this subunit as a scaffold protein within the polymerase holoenzyme. Bioseparation, 2000, 9(1), 37 - 41 Contamination of an anion-exchange membrane by glutathione; Gotoh T et al.; Electrodialysis, which can separate electrolytes under mild conditions by using ion-exchange membranes, is a strong candidate for separation of GSH from yeast extracts, because GSH is unstable and easily oxidized forming a disulfide bond especially under alkali conditions . In this paper, sorption behavior of GSH on an anion-exchange membrane, in the pH 3-6 region that is expected to be the most preferable for its electro-dialytic separation, was examined . Sorption of GSH on a Selemion-AMV anion-exchange membrane was accelerated as the pH of the membrane-contact solution increased, and there was a good correlation between the sorbed amounts and the molar fraction of monovalent anionic species of GSH . However, the amounts of GSH desorbed from the membrane by a NaCl desorbing solution were much lower than the initial sorbed amounts, and the difference between them was enlarged with increasing pH . The GSH which was lost could be recovered by the addition of DTT in the membrane-contact and desorbing solutions . Similar results were also obtained with Cys . We thus concluded that an anion-exchange membrane would be contaminated by thiol compounds, such as GSH and Cys, through oxidative binding of the thiol group with the membrane, the local OH- concentration in which was enhanced due to attraction by the positively charged anion-exchange membrane. Bioseparation, 2000, 9(1), 29 - 36 Expanded bed chromatography of proteins in small-diameter columns . II . Methods development and scale up; Ghose S et al.; The scaled down system developed in Part I of this series was further validated by using a 1-cm diameter column for method development studies for the separation of two model proteins, alcohol dehydrogenase and alpha-glucosidase, from unclarified yeast homogenate by hydrophobic interaction expanded bed chromatography based on the STREAMLINE matrix . The efficacy of solids removal and establishment of optimal binding and separation condition by stepwise elution were investigated . Equilibration of the EBA column and loading at high salt strengths affected the subsequent recovery of the two target proteins . Although good resolution between the target proteins could be achieved, peak tailing was found to be a consistent problem . The optimised separation protocol was scaled up 25-fold to a column diameter of 5.0 cm . The results were in good agreement with the run conducted in the 1-cm column, indicating the potential of using the small columns as an viable approach for method scouting and development studies. Biochem J, 2000 Jun 15, 348 Pt 3, 585 - 90 A new method for the selection of protein interactions in mammalian cells; Rojo-Niersbach E et al.; In the present study we present a new method that allows for the selection of protein interactions in mammalian cells . We have used this system to verify two interactions previously characterized in vitro . (1) The interaction between human TATA-binding protein 1 and nuclear factor kappaB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1alpha, a protein implicated in chromatin remodelling . We observe for the first time that these interactions also occur in vivo . One protein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue . Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes . The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway . In the human fibroblast cell line HT1080HPRT(-) (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium . This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable. Rev Bras Biol, 2000 Feb, 60(1), 45 - 52 Comparison of artificial diets for rearing Corcyra cephalonica (Stainton, 1865) (Lep., Pyralidae) for Trichogramma mass production; Bernardi EB et al.; The objective of this research was, based on biological studies, to determine and adequate diet for rearing Corcyra cephalonica (Stainton, 1865) in the laboratory so as to permit the rearing of this factitious host for Trichogramma mass production . The research was conducted at 25 +/- 1 degrees C, RH 60 +/- 10% and photophase of 14 hours . Six artificial diets were compared: a) whole wheat flour (48.5%), ground rice (48.5%) and sugar (3%); b) ground rice (97%) and sugar (3%); c) whole wheat flour (48.5%), rice flour (48.5%) an sugar (3%); d) whole wheat flour (97%) and yeast (3%); e) wheat germ (97%) and yeast (3%); f) rice bran (94%), sugar (3%) and yeast (3%); f) rice bran (94%), sugar (3%) and yeast (3%) . All of the diets studied permitted the development of C . cephalonica although the diets with wheat germ and yeast and that consisting of rice bran, sugar and yeast proved to be the most adequate for rearing the moth . These diets reduced the total (egg-adult) cycle, shortened the egg laying period, and produced heavier adults . Studies on the fertility life tables showed that higher net reproduction rates (Ro) and finite ratio of increase (lambda) were obtained from adults reared on these diets. J Biol Chem, 2000 Aug 18, 275(33), 25255 - 61 MCL-1S, a splicing variant of the antiapoptotic BCL-2 family member MCL-1, encodes a proapoptotic protein possessing only the BH3 domain; Bae J et al.; MCL-1 (myeloid cell leukemia-1) is an antiapoptotic BCL-2 family protein discovered as an early induction gene during myeloblastic leukemia cell differentiation . This survival protein has the BCL-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region . We identified a short splicing variant of the MCL-1 mRNA in the human placenta encoding a protein, termed MCL-1 short (MCL-1S), with an altered C terminus as compared with the full-length MCL-1 long (MCL-1L), leading to the loss of BH1, BH2, and the transmembrane domains . Analysis of the human MCL-1 gene indicated that MCL-1S results from the splicing out of exon 2 during mRNA processing . MCL-1S, unlike MCL-1L, does not interact with diverse proapoptotic BCL-2-related proteins in the yeast two-hybrid system . In contrast, MCL-1S dimerizes with MCL-1L in the yeast assay and coprecipitates with MCL-1L in transfected mammalian cells . Overexpression of MCL-1S induces apoptosis in transfected Chinese hamster ovary cells, and the MCL-1S action was antagonized by the antiapoptotic MCL-1L . Thus, the naturally occurring MCL-1S variant represents a new proapoptotic BH3 domain-only protein capable of dimerizing with the antiapoptotic MCL-1L . The fate of MCL-1-expressing cells could be regulated through alternative splicing mechanisms and interactions of the resulting anti- and proapoptotic gene products. J Biol Chem, 2000 Aug 18, 275(33), 25700 - 10 Molecular anatomy of the peroxin Pex12p: ring finger domain is essential for Pex12p function and interacts with the peroxisome-targeting signal type 1-receptor Pex5p and a ring peroxin, Pex10p; Okumoto K et al.; The three peroxin genes, PEX12, PEX2, and PEX10, encode peroxisomal integral membrane proteins with RING finger at the C-terminal part and are responsible for human peroxisome biogenesis disorders . Mutation analysis in PEX12 of Chinese hamster ovary cell mutants revealed a homozygous nonsense mutation at residue Trp263Ter in ZP104 cells and a pair of heterozygous nonsense mutations, Trp170Ter and Trp114Ter, in ZP109 . This result and domain mapping of Pex12p showed that RING finger is essential for peroxisome-restoring activity of Pex12p but not necessary for targeting to peroxisomes . The N-terminal region of Pex12p, including amino acid residues at positions 17-76, was required for localization to peroxisomes, while the sequence 17-76 was not sufficient for peroxisomal targeting . Peroxins interacting with RING finger of Pex2p, Pex10p, and Pex12p were investigated by yeast two-hybrid as well as in vitro binding assays . The RING finger of Pex12p bound to Pex10p and the PTS1-receptor Pex5p . Pex10p also interacted with Pex2p and Pex5p in vitro . Moreover, Pex12p was co-immunoprecipitated with Pex10p from CHO-K1 cells, where Pex5p was not associated with the Pex12p-Pex10p complex . This observation suggested that Pex5p does not bind to, or only transiently interacts with, Pex10p and Pex12p when Pex10p and Pex12p are in the oligomeric complex in peroxisome membranes . Hence, the RING finger peroxins are most likely to be involved in Pex5p-mediated matrix protein import into peroxisomes. Curr Opin Plant Biol, 2000 Jun, 3(3), 205 - 10 Delivering copper within plant cells; Himelblau E et al.; Two genes recently identified in Arabidopsis thaliana may be involved in sequestering free copper ions in the cytoplasm and delivering copper to post-Golgi vesicles . The genes COPPER CHAPERONE and RESPONSIVE TO ANTAGONIST1 are homologous to copper-trafficking genes from yeast and humans . This plant copper-delivery pathway is required to create functional ethylene receptors . The pathway may also facilitate the transport of copper from senescing leaf tissue . In addition, several other genes have been identified recently that may have a role in copper salvage during senescence. Curr Opin Plant Biol, 2000 Jun, 3(3), 211 - 6 Phytochelatin biosynthesis and function in heavy-metal detoxification; Cobbett CS; Plants respond to heavy-metal toxicity via a number of mechanisms . One such mechanism involves the chelation of heavy metals by a family of peptide ligands, the phytochelatins . Molecular genetic approaches have resulted in important advances in our understanding of phytochelatin biosynthesis . In particular, genes encoding the enzyme phytochelatin synthase have been isolated from plant and yeast species . Unexpectedly, genes with similar sequences to those encoding phytochelatin synthase have been identified in some animal species. Exp Cell Res, 2000 Jun 15, 257(2), 272 - 80 The nuclear DEAD box RNA helicase p68 interacts with the nucleolar protein fibrillarin and colocalizes specifically in nascent nucleoli during telophase; Nicol SM et al.; The DEAD box protein, p68, is an established RNA-dependent ATPase and RNA helicase in vitro, but neither the physiological function of this protein nor the macromolecules with which it interacts are known . Using a yeast two-hybrid screen, we identified the nucleolar protein, fibrillarin, as a protein that interacts with p68 . Coimmunoprecipitation experiments confirmed that p68 and fibrillarin can form complexes in cellular extracts, and deletion analysis identified regions in each protein responsible for mediating the interaction . Immunofluorescence studies using confocal microscopy revealed that, in interphase cells, while fibrillarin is predominantly nucleolar, p68 shows a diffuse granular nuclear staining but is largely excluded from the nucleoli . Strikingly, both proteins colocalize in nascent nucleoli during late telophase . These data are consistent with a role for p68 either in postmitotic nucleolar reassembly or in the activation of ribosomal DNA transcription/preribosomal RNA processing during telophase and suggest that differential subnuclear compartmentalization may be a mechanism by which interaction of p68 with fibrillarin is regulated in the cell . J Mol Evol, 2000 Jun, 50(6), 497 - 509 Anopheles stephensi Dox-A2 shares common ancestry with genes from distant groups of eukaryotes encoding a 26S proteasome subunit and is in a conserved gene cluster; Garvey CF et al.; The sequence of a cloned Anopheles stephensi gene showed 72% inferred amino acid identity with Drosophila melanogaster Dox-A2 and 93% with its putative ortholog in Anopheles gambiae . Dox-A2 is the reported but herein disputed structural locus for diphenol oxidase A2 . Database searches identified Dox-A2 related gene sequences from 15 non-insect species from diverse groups . Phylogenetic trees based on alignments of inferred protein sequences, DNA, and protein motif searches and protein secondary structure predictions produced results consistent with expectations for genes that are orthologous . The only inconsistency was that the C-terminus appears to be more primitive in the yeasts than in plants . In mammals, plants, and yeast these genes have been shown to code for a non-ATPase subunit of the PA700 (19S) regulatory complex of 26S proteasome . The analyses indicated that the insect genes contain no divergent structural features, which taken within an appraisal of all available data, makes the reported alternative function highly improbable . A plausible additional role, in which the 26S proteasome is implicated in regulation of phenol oxidase, would also apply to at least the mammalian genes . No function has yet been reported for the other included sequences . These were from genome projects and included Caenorhabiditus elegans, Arabidopsis thaliana, Fugu rubripes, and Toxoplasma gondii . A consensus of the results predicts a protein containing exceptionally long stretches of helix with a hydrophilic C-terminus . Phosphorylation site motifs were identified at two conserved positions . Possible SRY and GATA-1 binding motifs were found at conserved positions upstream of the mosquito genes . The location of A . stephensi Dox-A2 was determined by in situ hybridization at 34D on chromosome arm 3R . It is in a conserved gene cluster with respect to the other insects . However, the A . stephensi cluster contains a gene showing significant sequence identity to human and pigeon carnitine acetyltransferase genes, therefore showing divergence with the distal end of the D . melanogaster cluster. Genetics, 2000 Jun, 155(2), 803 - 12 Physical mapping of male fertility and meiotic drive quantitative trait loci in the mouse t complex using chromosome deficiencies; Planchart A et al.; The t complex spans 20 cM of the proximal region of mouse chromosome 17 . A variant form, the t haplotype (t), exists at significant frequencies in wild mouse populations and is characterized by the presence of inversions that suppress recombination with wild-type (+) chromosomes . Transmission ratio distortion and sterility are associated with t and affect males only . It is hypothesized that these phenomena are caused by trans-acting distorter/sterility factors that interact with a responder locus (Tcr(t)) and that the distorter and sterility factors are the same because homozygosity of the distorters causes male sterility . One factor, Tcd1, was previously shown to be amorphic using a chromosome deletion . To overcome limitations imposed by recombination suppression, we used a series of deletions within the t complex in trans to t chromosomes to characterize the Tcd1 region . We find that the distorter activity of Tcd1 is distinct from a linked sterility factor, originally called tcs1 . YACs mapped with respect to deletion breakpoints localize tcs1 to a 1.1-Mb interval flanked by D17Aus9 and Tctex1 . We present evidence for the existence of multiple proximal t complex regions that exhibit distorter activity . These studies demonstrate the utility of chromosome deletions for complex trait analysis. J Mol Biol, 2000 Jun 9, 299(3), 813 - 25 Mg(2+) binding to tRNA revisited: the nonlinear Poisson-Boltzmann model; Misra VK et al.; Our current understanding of Mg(2+) binding to RNA, in both thermodynamic and structural terms, is largely based on classical studies of transfer RNAs . Based on these studies, it is clear that magnesium ions are crucial for stabilizing the folded structure of tRNA . We present here a rigorous theoretical model based on the nonlinear Poisson-Boltzmann (NLPB) equation for understanding Mg(2+) binding to yeast tRNA(Phe) . We use this model to interpret a variety of experimental Mg(2+) binding data . In particular, we find that the NLPB equation provides a remarkably accurate description of both the overall stoichiometry and the free energy of Mg(2+) binding to yeast tRNA(Phe) without any fitted parameters . In addition, the model accurately describes the interaction of Mg(2+) with localized regions of the RNA as determined by the pK(a) shift of differently bound fluorophores . In each case, we find that the model also reproduces the univalent salt-dependence and the anticooperativity of Mg(2+) binding . Our results lead us to a thermodynamic description of Mg(2+) binding to yeast tRNA(Phe) based on the NLPB equation . In this model, Mg(2+) binding is simply explained by an ensemble of ions distributed according to a Boltzmann weighted average of the mean electrostatic potential around the RNA . It appears that the entire ensemble of electrostatically bound ions superficially mimics a few strongly coordinated ions . In this regard, we find that Mg(2+) stabilizes the tertiary structure of yeast tRNA(Phe) in part by accumulating in regions of high negative electrostatic potential . These regions of Mg(2+) localization correspond to bound ions that are observed in the X-ray crystallographic structures of yeast tRNA(Phe) . Based on our results and the available thermodynamic data, there is no evidence that specifically coordinated Mg ions have a significant role in stabilizing the native tertiary structure of yeast tRNA(Phe) in solution . J Mol Biol, 2000 Jun 9, 299(3), 667 - 80 Selection of homeotic proteins for binding to a human DNA replication origin; de Stanchina E et al.; We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene . Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence . All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators . We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene . The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays . In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells . We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation . Biotechnol Prog, 2000 May-Jun, 16(3), 435 - 41 Process options in hepatitis B surface antigen extraction from transgenic potato; Dogan B et al.; The process conditions for recombinant hepatitis B surface antigen (HBsAg) extraction from transgenic potato were examined . The effects of temperature, the reducing agent beta-mercaptoethanol (BME), and proteinase inhibitors on the level of antigenic activity of recovered HBsAg were determined . Sedimentation profiles were performed to characterize HBsAg assembly into virus-like particles . Increasing the temperature of the sample for about 1 min increased the measured HBsAg antigenic activity . The optimum temperature was around 50 degrees C . A 3-fold enhancement of the antigenic activity was obtained in extract from transgenic potato expressing HBsAg, when monoclonal antibodies were used to assay for HBsAg . When antigenic activity was determined by polyclonal antibodies, no enhancement in the antigenic activity was obtained . Temperature may affect the conformation of the a epitope to which the monoclonal antibodies bind or alter the fluidity of surface lipid regions . BME increased the antigenic activity of HBsAg up to 4-fold when monoclonal antibodies directed against the a determinant were used, but there was no increase with polyclonal antibodies . This observation suggests that BME affects the structure or presentation of the a epitope . In the presence of BME and leupeptin, a proteinase inhibitor, higher antigenic activity was obtained . Leupeptin might protect the antigen, which might become more susceptible to proteolytic degradation after reduction, as a result of stimulation of sulfhydryl proteases . Although both temperature and BME increased the antigenic activity of HBsAg individually, when combined their interaction was antagonistic, resulting in reduced antigenic activity . Different proteinase inhibitors, including leupeptin, aprotinin, E-64, pefabloc, and pepstatin, had no significant effect on HBsAg from potato extract in a 2 h period in the absence of BME . The sedimentation profile of potato-produced HBsAg was determined in 5-30% sucrose gradients . Yeast-derived recombinant HBsAg was used as a positive control . The HBsAg from transgenic potato showed sedimentation and density properties that are very similar to the yeast-produced antigen, indicating assembly into virus-like particles . BME treatment did not change the sedimentation profile. Biochem Biophys Res Commun, 2000 Jun 7, 272(2), 466 - 71 Interaction of Vesl-1L/Homer 1c with syntaxin 13; Minakami R et al.; Vesl-1S/Homer 1a, reported originally as Vesl/Homer, was isolated as a synaptic plasticity-regulated gene . The expression of Vesl-1S/Homer 1a is regulated during long-term potentiation in the hippocampus . Vesl-1L/Homer 1c, which appears to be formed by a splicing event, shares the N-terminus with Vesl-1S/Homer 1a and also contains additional amino acids at the C-terminus . The short form and the long form of the family members both interact with group 1 metabotropic glutamate receptors (mGluRs) . We herein report the identification of syntaxin 13 as a molecule that interacts with Vesl-1L using yeast two-hybrid screening . Syntaxin 13 is a member of the syntaxin family and is regarded as soluble N-ethylmaleimide-sensitive attachment proteins receptors (SNAREs) in the endosomal membranes . The interaction of Vesl-1L and syntaxin 13 was biochemically confirmed by in vitro binding assays . The coexpression of the two proteins in the transfected cells resulted in a colocalization in the intracellular vesicle-like structures . We thus propose that the association of Vesl-1L with syntaxin 13 plays a role in the translocation of Vesl-1L to the intracellular organelles . Biochem Biophys Res Commun, 2000 Jun 7, 272(2), 400 - 2 Identification of six CAG repeat domains into the human chromosomic region 12q24.1; Aguiar J; Six different domains of CAG repeats from a human chromosome 12-specific cosmid library were identified, cloned, and sequenced . These CAG repeat domains were localized into the human chromosomic region 12q24.1 . Five of them constitute repeat candidates for expansions in autosomal dominant neurological disorders with genetic anticipation, and they can also contribute to the chromosome walking in the human genome project . Mol Genet Metab, 2000 May, 70(1), 69 - 74 The tissue-specific, alternatively spliced single ATG exon of the type 3 voltage-dependent anion channel gene does not create a truncated protein isoform in vivo; Decker WK et al.; Voltage-dependent anion channels (VDACs) are small, integral membrane proteins that traverse the outer mitochondrial membrane and conduct ATP and other small metabolites . They are known to bind several kinases of intermediary metabolism in a tissue-specific fashion, have been found in close association with the adenine nucleotide translocator of the inner mitochondrial membrane, and are hypothesized to form part of the mitochondrial permeability transition pore, which results in the release of cytochrome c at the onset of apoptotic cell death . VDACs are found throughout all strata of eukaryotic evolution and exhibit biophysical characteristics that are well conserved from yeast to mammals . The mammalian VDAC gene family consists of three isoforms, each of which shares approximately 70% sequence identity with the other two family members . Recently, we reported that a single codon (ATG) exon is alternatively spliced into the transcript of the type 3 voltage-dependent anion channel (VDAC3) in a tissue-specific fashion . This unusual splicing event was shown to be conserved between mouse and human, and we theorized that the spliced exon could lead to the creation of an alternative translational initiation site . Here we report that a highly specific polyclonal VDAC3 antibody was unable to detect the truncated protein isoform indicative of this putative downstream start site . From these in vivo studies, we conclude that the alternatively spliced exon results in the insertion of a single methionine residue at amino acid position 39 of the mature VDAC3 protein . Additionally, we have used a cross-species genomic sequence comparison to identify conserved regions that may be involved in the regulation of small exon splicing . J Biochem (Tokyo), 2000 Jun, 127(6), 941 - 3 New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver; Tomisugi Y et al.; 20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast . To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source . Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them . The crystals belong to the same space group but have different cell dimensions . One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution . The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316 . 0 A, and diffracts to 4.0 A resolution . The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes. Microbiology, 2000 May, 146 ( Pt 5), 1045 - 51 Two distinct 18S rRNA secondary structures in Dipodascus (Hemiascomycetes); Ueda-Nishimura K et al.; The nucleotide sequences of the 18S rRNA gene from ascomycetous yeast-like fungi in the genera Dipodascus, Galactomyces and Geotrichum were determined and the tested strains were separated into two groups by sequence length . In group 1, the length and secondary structure of 18S rRNA corresponded to those of typical eukaryotes . In group 2, the 18S rRNA gene sequences were about 150 nt shorter than those of most other eukaryotes and the predicted secondary structure lacked helices 10 and E21-5 . Many substitutions and some deletions in group 2 18S rRNA gene were not only found in variable regions, but also in regions that are highly conserved among ascomycetes . Despite the considerable differences in 18S rRNA gene sequence and secondary structure between group 2 and other fungi, including group 1, phylogenetic analysis revealed that groups 1 and 2 are closely related . These findings suggest that a number of deletions occurred in the 18S rRNA of the common ancestor of group 2 strains. Gene, 2000 May 16, 249(1-2), 135 - 44 Site-specific integration of targeted DNA into animal cell genomes; Koch KS et al.; Novel genetically engineered retroviral vectors and targeting plasmids are described that enable the site-specific targeting of exogenous DNA into the genomes of cultured animal cells . The protocol involves the transduction of competent cells by a chimeric retroviral vector containing a transcription unit composed of two linked cassettes: an upstream marker gene under the control of the viral 5' LTR; and a downstream reporter trap containing a strong promoter 5' to a 48bp yeast FRT element . When cells containing such integrated units are co-transfected with a plasmid encoding yeast FLP recombinase and a promoterless targeting plasmid containing a reporter cDNA tract 3' to an homologous FRT element, the targeting plasmid recombines at the chromosomally preconfigured FRT site, and a new hemizygous function is introduced into the downstream cassette . These reagents provide a new portable system for site-specific targeting of chemically modified genes into uniform and unique sites in genomically integrated transcription units. J Inorg Biochem, 2000 Apr, 79(1-4), 11 - 9 Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein; Stillman MJ et al.; The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I) . Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein . CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures . Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data . Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I) . The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells . We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I) . Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM . Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described. Endocrinology, 2000 Jun, 141(6), 1977 - 88 Stat 5B, activated by insulin in a Jak-independent fashion, plays a role in glucokinase gene transcription; Sawka-Verhelle D et al.; Stat proteins are SH2 domain-containing transcription factors that are activated by various cytokines and growth factors . In a previous work, we have identified Stat 5B as a substrate of the insulin receptor based on yeast two-hybrid and mammalian cell transfection studies . In the present study, we have approached the biological relevance of the interaction between the insulin receptor and the transcription factor Stat 5B . Firstly, we show that both insulin and insulin-like growth factor I lead to tyrosine phosphorylation of Stat 5B, and this promotes binding of the transcription factor to the beta-casein promoter containing a Stat 5 binding site . Further, we demonstrate that insulin stimulates the transcriptional activity of Stat 5B . Activation of Stat 5B by insulin appears to be Jak2-independent, whereas Jak2 is required for GH-induced Stat 5B activation . Hence the pathway by which Stat 5B is activated by insulin is different from that used by GH . In addition, by using Jak1- and Tyk2-deficient cells we exclude the involvement of both Jak1 and Tyk2 in Stat 5B activation by insulin . Taken together, our results strengthen the notion that insulin receptor can directly activate Stat 5B . More importantly, we have identified a Stat 5 binding site in the human hepatic glucokinase promoter, and we show that insulin leads to a Stat 5B-dependent increase in transcription of a reporter gene carrying this promoter . These observations favor the idea that Stat 5B plays a role in mediating the expression of the glucokinase gene induced by insulin . As a whole, our results provide evidence for the occurrence of a newly identified circuit in insulin signaling in which the cell surface receptor is directly linked to nuclear events through a transcription factor . Further, we have revealed an insulin target gene whose expression is, at least in part, dependent on Stat 5B activation and/or binding. Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6350 - 5 A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing; Polydorides AD et al.; The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing . We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB) . The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons . brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei . We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor alpha2 (GlyRalpha2) pre-mRNA, and this binding is implicated in Nova-dependent regulation of splicing . Cotransfection assays with a GlyRalpha2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyRalpha2 E3A . brPTB binds to a 90-nt GlyRalpha2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova . When a putative binding site for brPTB on the GlyRalpha2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears . These results suggest that brPTB is a tissue-restricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons. J Biol Chem, 2000 Sep 1, 275(35), 27212 - 20 Fibrillarin genes encode both a conserved nucleolar protein and a novel small nucleolar RNA involved in ribosomal RNA methylation in Arabidopsis thaliana; Barneche F et al.; Fibrillarin is a key nucleolar protein in eukaryotes which associates with box C/D small nucleolar RNAs (snoRNAs) directing 2'-O-ribose methylation of the rRNA . In this study we describe two genes in Arabidopsis thaliana, AtFib1 and AtFib2, encoding nearly identical proteins conserved with eukaryotic fibrillarins . We demonstrate that AtFib1 and AtFib2 proteins are functional homologs of the yeast Nop1p (fibrillarin) and can rescue a yeast NOP1-null mutant strain . Surprisingly, for the first time in plants, we identified two isoforms of a novel box C/D snoRNA, U60.1f and U60.2f, nested in the fifth intron of AtFib1 and AtFib2 . Interestingly after gene duplication the host intronic sequences completely diverged, but the snoRNA was conserved, even in other crucifer fibrillarin genes . We show that the U60f snoRNAs accumulate in seedlings and that their targeted residue on the 25 S rRNA is methylated . Our data reveal that the three modes of expression of snoRNAs, single, polycistronic, and intronic, exist in plants and suggest that the mechanisms directing rRNA methylation, dependent on fibrillarin and box C/D snoRNAs, are evolutionarily conserved in plants. Cytogenet Cell Genet, 2000, 88(3-4), 289 - 95 Delineation and physical separation of novel translocation breakpoints on chromosome 1p in two genetically closely associated childhood tumors; Steenman MJ et al.; Sporadic childhood tumors associated with Beckwith-Wiedemann syndrome (BWS) all show abnormalities of the same region on chromosome 11 . In addition to chromosome 11, other chromosome regions are affected in some of these tumor types . In this study we analyzed the region on chromosome 1p involved in the etiology of BWS-associated tumors, Wilms tumor, rhabdomyosarcoma, and hepatoblastoma . For this purpose we determined the location of two novel translocation breakpoints in this chromosome region in cells from a Wilms tumor and cells from a rhabdomyosarcoma . We constructed a map of the region and found that both breakpoints are separated by at least 875 kb . We identified a PAC clone which crosses the rhabdomyosarcoma breakpoint and found several exons within this clone . We established that this breakpoint is located proximal to the PAX7 gene and, therefore, identified a new region involved in the etiology of rhabdomyosarcomas . Cytogenet Cell Genet, 2000, 88(3-4), 249 - 52 Chromosome mapping of the human genes encoding the MAP kinase kinase MEK1 (MAP2K1) to 15q21 and MEK2 (MAP2K2) to 7q32; Meloche S et al.; Activation of the ERK mitogen-activated protein (MAP) kinase pathway has been implicated in the regulation of cell growth, differentiation and senescence . In this pathway, the MAP kinases ERK1/ERK2 are phosphorylated and activated by the dual-specificity kinases MEK1 and MEK2, which in turn are activated by serine phosphorylation by a number of MAP kinase kinase kinases . We report here the chromosomal localization of the human genes encoding the MAP kinase kinase isoforms MEK1 and MEK2 . Using a combination of fluorescence in situ hybridization, somatic cell hybrid analysis, DNA sequencing and yeast artificial chromosome (YAC) clone analysis, we have mapped the MEK1 gene (MAP2K1) to chromosome 15q21 . We also present evidence for the presence of a MEK1 pseudogene on chromosome 8p21 . The MEK2 gene (MAP2K2) was mapped to chromosome 7q32 by fluorescence in situ hybridization and YAC clone analysis . Cytogenet Cell Genet, 2000, 88(3-4), 225 - 9 Expression map of human chromosome region 17p13.3, spanning the RP13 dominant retinitis pigmentosa locus, the Miller-Dieker lissencephaly syndrome (MDLS) region, and a putative tumour suppressor locus; McHale JC et al.; Chromosome region 17p13.3 is rich in genes, with 223 expressed sequence tags (ESTs) within the last 15 cM (7 Mb) of chromosome 17p in the GeneMap database . Loci for dominant retinitis pigmentosa (RP13), central areolar choroidal dystrophy (CACD), anterior polar cataract (CTAA2), Miller-Dieker lissencephaly syndrome (MDLS), and a region of tumour loss of heterozygosity (LOH) distinct from TP53 all map into the region adjacent to the 17p telomere . To date, however, there is no physical map of the region, which has resisted the efforts of the CEPH and Whitehead physical mapping programmes to generate contiguous clones across it . We have created a physical map covering approximately 3.5 Mb (6 cM)in this region, spanning the RP13 interval and extending distally to the gene MDCR (formerly, LIS1), which, when deleted, leads to the MDLS phenotype . The region covered is also the point of maximum LOH in lung cancer and has been implicated in the pathogenesis of many other human cancers . The map orders 47 sequence tagged sites, including 32 genes or ESTs, nine genetic markers, four anonymous sequences, and two YAC end clones, and highlights new candidate ESTs for involvement in RP13, MDLS, CTAA2, and a tumour-susceptibility gene . Hum Reprod, 2000 Jun, 15(6), 1289 - 94 Increased frequency of mutations in DNA from infertile men with meiotic arrest; Nudell D et al.; In diverse organisms from yeast to mice, mutations in numerous genes required for DNA repair may lead to defects in meiosis . Although it is likely that meiosis is conserved throughout evolution, little is known about the genetics of meiosis in humans even though meiotic arrest associated with azoospermia is common . In this work, we compared the sequence fidelity of a polymorphic marker amplified from DNA of two groups of patients: those with testis biopsy suggesting meiotic arrest and those with normal spermatogenesis who were obstructed . We demonstrated that mutations are more common in DNA from testicular tissue derived from men with meiotic arrest than in DNA from testicular tissue derived from men with normal spermatogenesis and physical obstruction (P < 0.05) . No mutations were observed in blood tissue from either group of men . This suggests the possibility that defects in genes required in DNA repair could contribute to meiotic arrest in men just as has been observed in other organisms. J Biol Chem, 2000 Jun 2, 275(22), 16827 - 36 Interaction of GRASP, a protein encoded by a novel retinoic acid-induced gene, with members of the cytohesin family of guanine nucleotide exchange factors; Nevrivy DJ et al.; A novel, retinoic acid-induced gene, GRP1-associated scaffold protein (GRASP), was isolated from P19 embryonal carcinoma cells using a subtractive screening strategy . GRASP was found to be highly expressed in brain and exhibited lower levels of expression in lung, heart, embryo, kidney, and ovary . The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may be a component of a larger protein complex in the cell . Although GRASP does not harbor a predicted membrane spanning domain(s), the protein was observed to be associated with the plasma membrane of transiently transfected mammalian cells . Yeast two-hybrid screening revealed that GRASP interacted strongly with the General Receptor for Phosphoinositides 1 (GRP1), a brefeldin A-insensitive guanine nucleotide exchange factor for the ADP-ribosylation factor family of proteins . GRASP . GRP1 interactions were also demonstrated in vitro and in mammalian cells in which GRASP was shown to enhance GRP1 association with the plasma membrane . Furthermore, GRASP colocalized with endogenous ADP-ribosylation factors at the plasma membrane in transfected cells, suggesting that GRASP may modulate signaling by this family of small GTPases. J Biol Chem, 2000 Aug 18, 275(33), 25820 - 30 Expression of human topoisomerase I with a partial deletion of the linker region yields monomeric and dimeric enzymes that respond differently to camptothecin; Ireton GC et al.; Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine . Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains . Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer . The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits . The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive . However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme . The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker . Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin. Am J Hum Genet, 2000 Jul, 67(1), 14 - 22 Epub 2000 May 25. Additional copies of the proteolipid protein gene causing Pelizaeus-Merzbacher disease arise by separate integration into the X chromosome; Hodes ME et al.; The proteolipid protein gene (PLP) is normally present at chromosome Xq22 . Mutations and duplications of this gene are associated with Pelizaeus-Merzbacher disease (PMD) . Here we describe two new families in which males affected with PMD were found to have a copy of PLP on the short arm of the X chromosome, in addition to a normal copy on Xq22 . In the first family, the extra copy was first detected by the presence of heterozygosity of the AhaII dimorphism within the PLP gene . The results of FISH analysis showed an additional copy of PLP in Xp22.1, although no chromosomal rearrangements could be detected by standard karyotype analysis . Another three affected males from the family had similar findings . In a second unrelated family with signs of PMD, cytogenetic analysis showed a pericentric inversion of the X chromosome . In the inv(X) carried by several affected family members, FISH showed PLP signals at Xp11.4 and Xq22 . A third family has previously been reported, in which affected members had an extra copy of the PLP gene detected at Xq26 in a chromosome with an otherwise normal banding pattern . The identification of three separate families in which PLP is duplicated at a noncontiguous site suggests that such duplications could be a relatively common but previously undetected cause of genetic disorders. J Biol Chem, 2000 Aug 18, 275(33), 25411 - 7 NIPP1-mediated interaction of protein phosphatase-1 with CDC5L, a regulator of pre-mRNA splicing and mitotic entry; Boudrez A et al.; NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles . We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module . A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G(2)/M progression and pre-mRNA splicing . CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells . Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments . The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g . by cyclin E-Cdk2 . When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G(2)/M transition . However, the FHA domain blocked beta-globin pre-mRNA splicing in nuclear extracts . A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects . We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 337 - 46 Modestobacter multiseptatus gen . nov., sp . nov., a budding actinomycete from soils of the Asgard Range (Transantarctic Mountains); Mevs U et al.; Oligotrophic PYGV medium, inoculated with soils from Linnaeus Terrace (1600 m, Antarctica), yielded four aerobic actinomycetes with short rods, multiple and irregular septa and often motile buds . Cells were 1.0-2.8 x 1.0-3.0 microm and colonies were beige to pink . The isolates were nearly identical in physiological and biochemical tests . Three strains grew from 0 degrees C to 25-28 degrees C, but one was psychrophilic with a maximum growth temperature of 20 degrees C . Carbon sources utilized were D-glucose, D-galactose, lactose, sucrose or mannitol; malate, succinate, fumarate, pyruvate or glutarate were decarboxylated aerobically . Peptone and yeast extract were the preferred nitrogen sources . Nitrate was reduced aerobically or anaerobically . Cell walls contained meso-diaminopimelic acid, glutamate, alanine, glycine, galactose, glucose and ribose . Major fatty acids of strains AA-802, -824, -825 and -826T were n18:1, i16:0 and ai17:0 . Major respiratory quinones were MK-9(H4) and MK-8(H4) . Polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol . Phosphatidylglycerol was found in most strains . The DNA G+C contents were 68-70 mol% . In 16S rDNA analyses, similarity values obtained for 500 nucleotides from the 5' terminus were > 99.5% . Almost complete sequences from AA-826T and -825 were 99.9% similar . Strain AA-826T belonged to a novel cluster of desert soil and rock isolates within the Geodermatophilaceae and was equidistantly related to members of Geodermatophilus and to a Blastococcus lineage . The four isolates represent a new genus, Modestobacter gen . nov., with Modestobacter multiseptatus sp . nov . as the type species . The type strain, Modestobacter multiseptatus AA-826T, was deposited in the DSMZ as DSM 44406T. Eur J Cell Biol, 2000 Apr, 79(4), 240 - 51 Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus; Behrend L et al.; Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis . We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules . Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation . In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage . The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis. J Cell Sci, 2000 Jun, 113 ( Pt 12), 2273 - 84 Hrs interacts with SNAP-25 and regulates Ca(2+)-dependent exocytosis; Kwong J et al.; Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane protein essential for synaptic vesicle exocytosis . To investigate the mechanisms by which SNAP-25 mediates neurosecretion, we performed a search for proteins that interact with SNAP-25 using a yeast two-hybrid screen . Here, we report the isolation and characterization of a SNAP-25-interacting protein that is the rat homologue of mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) . Hrs specifically interacts with SNAP-25, but not SNAP-23/syndet . The association of Hrs and SNAP-25 is mediated via coiled-coil interactions . Using an Hrs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in most brain regions . Subcellular fractionation studies demonstrate that in brain, Hrs exists in both cytosolic and membrane-associated pools . Studies using indirect immunofluorescence and confocal microscopy reveal that, in addition to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differentiated PC12 cells . Moreover, overexpression of Hrs in PC12 cells inhibits Ca(2+)-dependent exocytosis . These results suggest that Hrs is involved in regulation of neurosecretion through interaction with SNAP-25. J Cell Sci, 2000 Jun, 113 ( Pt 12), 2221 - 31 Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription; Javed A et al.; The Runt related transcription factors RUNX (AML/CBF(alpha)/PEBP2(alpha)) are key regulators of hematopoiesis and osteogenesis . Using co-transfection experiments with four natural promoters, including those of the osteocalcin (OC), multi drug resistance (MDR), Rous Sarcoma Virus long terminal repeat (LTR), and bone sialoprotein (BSP) genes, we show that each of these promoters responds differently to the forced expression of RUNX proteins . However, the three RUNX subtypes (i.e . AML1, AML2, and AML3) regulate each promoter in a similar manner . Although the OC promoter is activated in a C terminus dependent manner, the MDR, LTR and BSP promoters are repressed by three distinct mechanisms, either independent of or involving the AML C terminus, or requiring only the conserved C-terminal pentapeptide VWRPY . Using yeast two hybrid assays we find that the C terminus of AML1 interacts with a Groucho/TLE/R-esp repressor protein . Co-expression assays reveal that TLE proteins repress AML dependent activation of OC gene transcription . Western and northern blot analyses suggest that TLE expression is regulated reciprocally with the levels of OC gene expression during osteoblast differentiation . Digital immunofluorescence microscopy results show that TLE1 and TLE2 are both associated with the nuclear matrix, and that a significant subset of each colocalizes with AML transcription factors . This co-localization of TLE and AML proteins is lost upon removing the C terminus of AML family members . Our findings indicate that suppression of AML-dependent gene activation by TLE proteins involves functional interactions with the C terminus of AML at the nuclear matrix in situ . Our data are consistent with the concept that the C termini of AML proteins support activation or repression of cell-type specific genes depending on the regulatory organization of the target promoter and subnuclear localization. Mol Cell Biol, 2000 Jun, 20(12), 4462 - 73 The oncoprotein kinase chaperone CDC37 functions as an oncogene in mice and collaborates with both c-myc and cyclin D1 in transformation of multiple tissues; Stepanova L et al.; CDC37 encodes a 50-kDa protein that targets intrinsically unstable oncoprotein kinases including Cdk4, Raf-1, and v-src to the molecular chaperone Hsp90, an interaction that is thought to be important for the establishment of signaling pathways . CDC37 is required for proliferation in budding yeast and is coexpressed with cyclin D1 in proliferative zones during mouse development, a finding consistent with a positive role in cell proliferation . CDC37 expression may not only be required to support proliferation in cells that are developmentally programmed to proliferate but may also be required in cells that are inappropriately induced to initiate proliferation by oncogenes . Here we report that mouse mammary tumor virus (MMTV)-CDC37 transgenic mice develop mammary gland tumors at a rate comparable to that observed previously in MMTV-cyclin D1 mice . Moreover, CDC37 was found to collaborate with MMTV-c-myc in the transformation of multiple tissues, including mammary and salivary glands in females and testis in males, and also collaborates with cyclin D1 to transform the female mammary gland . These data indicate that CDC37 can function as an oncogene in mice and suggests that the establishment of protein kinase pathways mediated by Cdc37-Hsp90 can be a rate-limiting event in epithelial cell transformation. Mol Cell Biol, 2000 Jun, 20(12), 4350 - 8 Artificial recruitment of TFIID, but not RNA polymerase II holoenzyme, activates transcription in mammalian cells; Dorris DR et al.; In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain . Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells . Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters . In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains . In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter . Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters . Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme. J Biol Chem, 2000 Sep 15, 275(37), 28785 - 92 HPC3 is a new human polycomb orthologue that interacts and associates with RING1 and Bmi1 and has transcriptional repression properties; Bardos JI et al.; Polycomb group (PcG) proteins were first described in Drosophila as factors responsible for maintaining the transcriptionally repressed state of Hox/homeotic genes in a stable and heritable manner throughout development . A growing number of vertebrate genes related to the Drosophila PcG proteins have recently been identified, including two Polycomb orthologues, Pc2 and M33 . PcG proteins form multiprotein complexes, termed PcG bodies, that are thought to repress transcription by altering chromatin structure . Here we report the identification and characterization of HPC3 (human Polycomb 3), a novel PcG protein isolated in a yeast two-hybrid screen using human RING1 as bait . HPC3 shows strong sequence similarity to Drosophila Pc and also to vertebrate Pc2 and M33, particularly within the chromodomain and C-box . Previous studies indicate that M33 and human Pc2 (HPC2) can interact with RING1, and we show here that HPC3 also binds to RING1 . This interaction is dependent upon the HPC3 C-box but, only partially on the RING finger of RING1 . In contrast to HPC2, HPC3 interactions with RING1 are only observed in vivo with covalently modified forms of RING1 . HPC3 also colocalizes with other PcG proteins in human PcG bodies . Consistent with its role as a PcG member, HPC3 is able to act as a long range transcriptional silencer when targeted to a reporter gene by a heterologous DNA-binding domain . Taken together, these data suggest that HPC3 is part of a large multiprotein complex that also contains other PcG proteins and is involved in repression of transcriptional activity. J Biol Chem, 2000 Aug 4, 275(31), 23666 - 73 Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice; Oshel KM et al.; We have previously demonstrated that the important cis-acting elements regulating transcription of the human GLUT4 gene reside within 895 base pairs (bp) upstream of the transcription initiation site (Thai, M . V., Guruswamy, S., Cao, K . T., Pessin, J . E., and Olson, A . L . (1998) J . Biol . Chem . 273, 14285-14292) . Our studies demonstrated that an MEF2 binding site within this region was necessary, but not sufficient, for GLUT4 promoter function in transgenic mice . We have identified a second regulatory element (Domain I) that functions cooperatively with the MEF2 domain in regulating GLUT4 transcription . Using a yeast-one hybrid screen, we obtained a partial cDNA and generated an antibody directed against a protein binding specifically to Domain I . Sequence analysis of the partial cDNA indicates that the protein binding to Domain I is a novel protein . The antibody specifically labels two proteins of approximately 70 and 50 kDa in Western blot analysis . These molecular masses correspond to Domain I binding proteins identified by UV-cross-linking nuclear extracts to a Domain I probe . The antibody raised against the Domain I binding protein inhibited formation of a Domain I-protein complex in electrophoretic mobility shift assays . We conclude that we have identified an authentic, novel, Domain I binding protein required for transcriptional regulation of the human GLUT4 promoter. J Biol Chem, 2000 Aug 25, 275(34), 26172 - 7 Functional cloning and characterization of a novel nonhomeodomain protein that inhibits the binding of PBX1-HOX complexes to DNA; Abramovich C et al.; PBX1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during developmental and/or differentiation processes . A yeast two-hybrid screen of a fetal liver-hematopoietic cDNA library using PBX1a as bait led to the discovery of a novel non-homeodomain-containing protein that interacts with PBX1 as well as PBX2 and PBX3 . RNA analysis revealed it to be expressed in CD34(+) hematopoietic cell populations enriched in primitive progenitors, as is PBX1; search of the expressed sequence tag data base indicated that it is also expressed in other early embryonic as well as adult tissues . The full-length cDNA encodes a 731-amino acid protein that has no significant homology to known proteins . This protein that we have termed hematopoietic PBX-interacting protein (HPIP) is mainly localized in the cytosol and in small amounts in the nucleus . The region of PBX that interacts with HPIP includes both the homeodomain and immediate N-terminal flanking sequences . Strikingly, electrophoretic mobility shift assays revealed that HPIP inhibits the ability of PBX-HOX heterodimers to bind to target sequences . Moreover, HPIP strongly inhibits the transactivation activity of E2A-PBX . Together these findings suggest that HPIP is a new regulator of PBX function. J Biol Chem, 2000 Sep 1, 275(35), 27084 - 93 The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM; Hawkins CJ et al.; The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death . All caspases studied to date cleave substrates COOH-terminal to an aspartate . Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate . DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate . DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate . Expression of a dominant negative form of DRONC blocks cell death induced by the Drosophila cell death activators reaper, hid, and grim, and DRONC overexpression in flies promotes cell death . Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM . These observations suggest that DRONC acts to promote cell death . However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35 . We discuss possible models for DRONC function as a cell death inhibitor. Genes Chromosomes Cancer, 2000 Jun, 28(2), 153 - 63 A consistent pattern of RIN1 rearrangements in oral squamous cell carcinoma cell lines supports a breakage-fusion-bridge cycle model for 11q13 amplification; Shuster MI et al.; Gene amplification is a common feature of tumors . Overexpression of some amplified genes plays a role in tumor progression . Gene amplification can occur either extrachromosomally as double-minute chromosomes (dmin) or intrachromosomally in the form of homogeneously staining regions (hsrs) . Approximately one-half of our oral squamous cell carcinomas (OSCCs) are characterized by amplification of band 11q13, usually as an hsr located entopically (occurring or situated at the normal chromosomal site, as opposed to ectopically) . Using chromosomal fluorescence in situ hybridization (FISH), we confirmed the amplification of the cyclin D1 (CCND1/PRAD1) and fibroblast growth factor types 3 and 4 (FGF3/INT2 and FGF4/HSTF1) genes within the 11q13