Nat Genet, 2001 Mar, 27(3), 271 - 6 Recombinational DNA double-strand breaks in mice precede synapsis; Mahadevaiah SK et al.; In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis . Here we use an antibody specific for a phosphorylated histone (gamma-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse . We show that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene . Loss of gamma-H2AX staining (which in irradiated somatic cells is temporally linked with DSB repair) is temporally and spatially correlated with synapsis, even when this synapsis is 'non-homologous'. Nature, 2001 Mar 8, 410(6825), 227 - 30 Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans; Tissenbaum HA et al.; In Caenorhabditis elegans, mutations that reduce the activity of an insulin-like receptor (daf-2) or a phosphatidylinositol-3-OH kinase (age-1) favour entry into the dauer state during larval development and extend lifespan in adults . Downregulation of this pathway activates a forkhead transcription factor (daf-16), which may regulate targets that promote dauer formation in larvae and stress resistance and longevity in adults . In yeast, the SIR2 gene determines the lifespan of mother cells, and adding an extra copy of SIR2 extends lifespan . Sir2 mediates chromatin silencing through a histone deacetylase activity that depends on NAD (nicotinamide adenine dinucleotide) as a cofactor . We have surveyed the lifespan of C . elegans strains containing duplications of chromosomal regions . Here we report that a duplication containing sir-2.1-the C . elegans gene most homologous to yeast SIR2-confers a lifespan that is extended by up to 50% . Genetic analysis indicates that the sir-2.1 transgene functions upstream of daf-16 in the insulin-like signalling pathway . Our findings suggest that Sir2 proteins may couple longevity to nutrient availability in many eukaryotic organisms. Int J Cancer, 2001 Jan 20, 95(1), 23 - 8 TAP1 down-regulation in primary melanoma lesions: an independent marker of poor prognosis; Kamarashev J et al.; Melanoma tumor thickness is a major prognostic factor . Thin lesions, however, may metastasize, and sometimes thick tumors may not . To investigate the role of HLA class I-mediated antigen presentation, we correlated the expression of components of the antigen-processing machinery in primary melanoma lesions with their thickness and with the development of metastases . Seventeen formalin-fixed, paraffin-embedded primary melanomas thinner than 0.76 mm and 21 thicker than 1.50 mm were stained with anti-LMP2, -LMP7, -TAP1, -TAP2, -HLA class I and -beta2-microglobulin monoclonal antibodies . Twenty patients remained tumor-free in the follow-up period (10.5 +/- 1.8 years) . Eighteen patients relapsed within a median period of 15.0 months following tumor excision . Expression of all markers in the tested lesions was down-regulated, the frequency ranging from about 40% for LMP and TAP subunits to about 70% for HLA class I antigens . Expression of all markers was not correlated with tumor thickness . Only TAP1 and TAP2 down-regulation was significantly (p = 0.026 and 0.042, respectively) correlated with the development of metastases . This correlation was independent of tumor thickness for TAP1 . We suggest that TAP1 and probably TAP2 expression in primary lesions represents an independent prognostic marker in melanoma . Abnormalities in antigen presentation may account for the lack of absolute correlation between tumor thickness and prognosis . Biopolymers, 2000, 55(5), 407 - 14 Engineering of the hydrophobic core of an alpha-helical coiled coil; Kiyokawa T et al.; The amino acid sequence that forms the alpha-helical coiled coil structure has a representative heptad repeat denoted by defgabc, according to their positions . Although the a and d positions are usually occupied by hydrophobic residues, hydrophilic residues at these positions sometimes play important roles in natural proteins . We have manipulated a few amino acids at the a and d positions of a de novo designed trimeric coiled coil to confer new functions to the peptides . The IZ peptide, which has four heptad repeats and forms a parallel triple-stranded coiled coil, has Ile at all of the a and d positions . We show three examples: (1) the substitution of one Ile at either the a or d position with Glu caused the peptide to become pH sensitive; (2) the metal ion induced alpha-helical bundles were formed by substitutions with two His residues at the d and a positions for a medium metal ion, and with one Cys residue at the a position for a soft metal ion; and (3) the AAB-type heterotrimeric alpha-helical bundle formation was accomplished by a combination of Ala and Trp residues at the a positions of different peptide chains . Furthermore, we applied these procedures to prepare an ABC-type heterotrimeric alpha-helical bundle and a metal ion-induced heterotrimeric alpha-helical bundle. Mutat Res, 2001 Mar 1, 474(1-2), 93 - 103 Activation of Ty transposition by mutagens; Staleva Staleva L et al.; The induction of Ty1 transposition by mutagens (MMS and 4NQO) in asynchronous cultures and cells blocked in G1 and G2/M suggested G1 dependence of activation of Ty1 element by DNA damage . Northern blot analysis revealed immediate five-fold increase in levels of Ty1 transcript after 20min incubation of cells with 1 microg/ml 4NQO and four-fold increase in Ty1 RNA after treatment the cells with 0.1% MMS . Western blot analysis showed no difference in TyA protein in treated and untreated with mutagen cells . Quantitative mutagenicity assay and Northern blot analysis demonstrated dependence of induction of Ty1 element by DNA-damaging agents on the function of RAD9 gene and independence on DUN1 gene. Biol Psychiatry, 2001 Feb 15, 49(4), 333 - 9 Haplotype study of three polymorphisms at the dopamine transporter locus confirm linkage to attention-deficit/hyperactivity disorder; Barr CL et al.; BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is often treated using methylphenidate, a psychostimulant that inhibits the dopamine transporter . This led E.H . Cook and colleagues to consider the dopamine transporter locus (DAT1) as a primary candidate gene for ADHD . That group reported a significant association between ADHD and the 480-base pair (bp) allele of the variable number of tandem repeats (VNTR) polymorphism located in the 3' untranslated region of the DAT1 gene . This association was later replicated in additional studies . METHODS: The DAT1 gene has additional common polymorphisms in intron 9 and exon 9 . We investigated the possibility of linkage of DAT1 and ADHD using the VNTR polymorphism and two additional common polymorphisms in 102 nuclear families with an ADHD proband . Using the transmission disequilibrium test, we examined the transmission of the alleles of each of these polymorphisms, as well as the haplotypes of the polymorphisms . RESULTS: We did not observe significant evidence for the biased transmission of the alleles of either the VNTR or the additional two polymorphisms when examined individually, although there was a trend for the biased transmission of the 480-bp allele of the VNTR . When we examined the haplotypes of the three polymorphisms we found significant evidence for biased transmission of one of the haplotypes containing the 480-bp VNTR allele . We also genotyped six additional DNA sequence variants of the DAT1 gene . However, these variants were not sufficiently polymorphic in our sample to be informative . Two of the DNA variants that result in an amino acid change, Ala559Val and Glu602Gly, were not observed in our sample . CONCLUSIONS: Our results support previous findings of an association between the DAT1 gene and ADHD. Mol Cell, 2001 Feb, 7(2), 433 - 42 TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport; Sacher M et al.; TRAPP is a conserved protein complex required early in the secretory pathway . Here, we report two forms of TRAPP, TRAPP I and TRAPP II, that mediate different transport events . Using chemically pure TRAPP I and COPII vesicles, we have reconstituted vesicle targeting in vitro . The binding of COPII vesicles to TRAPP I is specific, blocked by GTPgammaS, and, surprisingly, does not require other tethering factors . Our findings imply that TRAPP I is the receptor on the Golgi for COPII vesicles . Once the vesicle binds to TRAPP I, the small GTP binding protein Ypt1p is activated and other tethering factors are recruited. Mol Cell, 2001 Feb, 7(2), 319 - 29 A biochemical function for the Sm complex; Zhang D et al.; Within the yeast commitment complex, SmB, SmD1, and SmD3 make direct contact with the pre-mRNA substrate, close to the 5' splice site . Only these three Sm proteins have long and highly charged C-terminal tails, in metazoa as well as in yeast . We replaced these proteins with tail-truncated versions . Genetic assays demonstrate that the tails contribute to similar and overlapping functions, and cross-linking assays show that the tails make direct contact with the pre-mRNA in a largely sequence-independent manner . Other biochemical assays indicate that they function at least in part to stabilize the U1 snRNP-pre-mRNA interaction . We speculate that this role may be general, and may have even evolved to aid weak intermolecular nucleic acid interactions of only a few base pairs. Cell, 2001 Feb 9, 104(3), 387 - 96 Cdc13 delivers separate complexes to the telomere for end protection and replication; Pennock E et al.; In Saccharomyces cerevisiae, the telomere binding protein Cdc13 mediates telomere replication by recruiting telomerase, and also performs an essential function in chromosome end protection . We show here that delivery of the Stn1 protein to the telomere, by fusing the DNA binding domain of Cdc13 (DBD(CDC13)) to Stn1, is sufficient to rescue the lethality of a cdc13 null strain and, hence, provide end protection . Telomere replication is still defective in this strain, but can be restored by delivering telomerase to the telomere as a DBD(CDC13)-telomerase fusion . These results establish Stn1 as the primary effector of chromosome end protection, whereas the principal function of Cdc13 is to provide a loading platform to recruit complexes that provide end protection and telomere replication. Nucleic Acids Res . 2001 Mar 15;29(6):E32. Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning; Noskov VN et al.; The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes . The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest . The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest . To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region . For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning . When vectors with a specific hook that was >/=60 bp were utilized, approximately 2% of transformants contained circular YACs with the Tg.AC transgene sequences . Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less . Thus, the minimal length of a unique sequence required for gene isolation by TAR is approximately 60 bp . No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning. Nucleic Acids Res, 2001 Mar 15, 29(6), 1341 - 51 Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells; Balajee AS et al.; Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is involved in DNA replication as well as in diverse DNA repair pathways . In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases delta and epsilon . In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques . Exposure of normal cells to gamma-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40-45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling . The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h . The PCNA foci formed after gamma-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion . Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after gamma-irradiation . We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks . The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells . Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks . Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks. Nucleic Acids Res, 2001 Mar 15, 29(6), 1300 - 7 Human securin, hPTTG, is associated with Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase; Romero F et al.; We have previously isolated the hpttg proto-oncogene, which is expressed in normal tissues containing proliferating cells and in several kinds of tumors . In fact, expression of hPTTG correlates with cell proliferation in a cell cycle-dependent manner . Recently it was reported that PTTG is a vertebrate analog of the yeast securins Pds1 and Cut2, which are involved in sister chromatid separation . Here we show that hPTTG binds to Ku, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) . hPTTG and Ku associate both in vitro and in vivo and the DNA-PK catalytic subunit phosphorylates hPTTG in vitro . Furthermore, DNA double-strand breaks prevent hPTTG-Ku association and disrupt the hPTTG-Ku complexes, indicating that genome damaging events, which result in the induction of pathways that activate DNA repair mechanisms and halt cell cycle progression, might inhibit hPTTG-Ku interaction in vivo . We propose that hPTTG might connect DNA damage-response pathways with sister chromatid separation, delaying the onset of mitosis while DNA repair occurs. Mol Cell Biol, 2001 Mar, 21(6), 2184 - 91 Distinct functional domains of nibrin mediate Mre11 binding, focus formation, and nuclear localization; Desai-Mehta A et al.; The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in the NBS1 gene, which encodes the protein nibrin . Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50 . Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair . In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic . In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts . Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells . However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci . Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50 . Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci . These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation . However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation . Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction. Mol Cell Biol, 2001 Mar, 21(6), 2085 - 97 The neuron-restrictive silencer element-neuron-restrictive silencer factor system regulates basal and endothelin 1-inducible atrial natriuretic peptide gene expression in ventricular myocytes; Kuwahara K et al.; Induction of the atrial natriuretic peptide (ANP) gene is a common feature of ventricular hypertrophy . A number of cis-acting enhancer elements for several transcriptional activators have been shown to play central roles in the regulation of ANP gene expression, but much less is known about contributions made by transcriptional repressors . The neuron-restrictive silencer element (NRSE), also known as repressor element 1, mediates repression of neuronal gene expression in nonneuronal cells . We found that NRSE, which is located in the 3' untranslated region of the ANP gene, mediated repression of ANP promoter activity in ventricular myocytes and was also involved in the endothelin 1-induced increase in ANP gene transcription . The repression was conferred by a repressor protein, neuron-restrictive silencer factor (NRSF) . NRSF associated with the transcriptional corepressor mSin3 and formed a complex with histone deacetylase (HDAC) in ventricular myocytes . Trichostatin A (TSA), a specific HDAC inhibitor, relieved NRSE-mediated repression of ANP promoter activity, and chromatin immunoprecipitation assays revealed the involvement of histone deacetylation in NRSE-mediated repression of ANP gene expression . Furthermore, in myocytes infected with recombinant adenovirus expressing a dominant-negative form of NRSF, the basal level of endogenous ANP gene expression was increased and a TSA-induced increase in ANP gene expression was apparently attenuated, compared with those in myocytes infected with control adenovirus . Our findings show that an NRSE-NRSF system plays a key role in the regulation of ANP gene expression by HDAC in ventricular myocytes and provide a new insight into the role of the NRSE-NRSF system outside the nervous system. Mol Cell Biol, 2001 Mar, 21(6), 2048 - 56 Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break; Signon L et al.; Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR) . In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion . Previously, we have shown that in the absence of RAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR . We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as with rad51Delta cells . DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1 (RDH54), SRS2, or SGS1 is deleted . Various double mutations largely eliminate both gene conversion and BIR, including rad51Delta rad50Delta, rad51Delta rad59Delta, and rad54Delta tid1Delta . These results demonstrate that there is a RAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumably MRE11 and XRS2 . The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms. Mol Cell Biol, 2001 Mar, 21(6), 2026 - 37 Fip1 regulates the activity of Poly(A) polymerase through multiple interactions; Helmling S et al.; Fip1 is an essential component of the Saccharomyces cerevisiae polyadenylation machinery and the only protein known to interact directly with poly(A) polymerase (Pap1) . Its association with Pap1 inhibits the extension of an oligo(A) primer by limiting access of the RNA substrate to the C-terminal RNA binding domain (C-RBD) of Pap1 . We present here the identification of separate functional domains of Fip1 . Amino acids 80 to 105 are required for binding to Pap1 and for the inhibition of Pap1 activity . This region is also essential for viability, suggesting that Fip1-mediated repression of Pap1 has a crucial physiological function . Amino acids 206 to 220 of Fip1 are needed for the interaction with the Yth1 subunit of the complex and for specific polyadenylation of the cleaved mRNA precursor . A third domain within amino acids 105 to 206 helps to limit RNA binding at the C-RBD of Pap1 . Our data demonstrate that the C terminus of Fip1 is required to relieve the Fip1-mediated repression of Pap1 in specific polyadenylation . In the absence of this domain, Pap1 remains in an inhibited state . These findings show that Fip1 has a crucial regulatory function in the polyadenylation reaction by controlling the activity of poly(A) tail synthesis through multiple interactions within the polyadenylation complex. Mol Cell Biol, 2001 Mar, 21(6), 1997 - 2007 Dosage suppressors of pds1 implicate ubiquitin-associated domains in checkpoint control; Clarke DJ et al.; In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p . Analysis of pds1 mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints . Though some components of these pathways are known, others remain to be identified . Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated . To identify loci that genetically interact with PDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37 degrees C . Genetic and functional interactions of two suppressors are described . RAD23 and DDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128 . rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints . Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls . Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128 . UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them . Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling . Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation. Mol Cell Biol, 2001 Mar, 21(5), 1819 - 27 Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events; Chen Q et al.; Yeast cells can survive in the absence of telomerase RNA, TLC1, by recombination-mediated telomere elongation . Two types of survivors, type I and type II, can be distinguished by their characteristic telomere patterns . RAD52 is essential for the generation of both types of survivors . Deletion of both RAD50 and RAD51 produces a phenotype similar to that produced by deletion of RAD52 . Here we examined the effects of the RAD50 and the RAD51 epistasis groups as well as the RAD52 homologue, RAD59, on the types of survivors generated in the absence of telomerase . rad59 mutations completely abolished the ability to generate type II survivors, while rad50 mutations decreased the growth viability of type II survivors but did not completely eliminate their appearance . Mutations in RAD51, RAD54, and RAD57 had the converse affect: they eliminated the ability of cells to generate type I survivors in a tlc1 strain . The triple mutant, tlc1 rad51 rad59, was not able to generate survivors . Thus either type I or type II recombination pathways can allow cells to survive in the absence of telomerase; however, elimination of both pathways in a telomerase mutant leads to the inability to elongate telomeres and ultimately cell death. Mol Cell Biol, 2001 Mar, 21(5), 1784 - 94 CAC3(MSI1) suppression of RAS2(G19V) is independent of chromatin assembly factor I and mediated by NPR1; Johnston SD et al.; Cac3p/Msi1p, the Saccharomyces cerevisiae homolog of retinoblastoma-associated protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF-I), a complex that assembles histones H3 and H4 onto replicated DNA . CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transduction pathway by an unknown mechanism . We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of either CAC1 or CAC2, subunits required for CAF-I function . CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function . Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm . In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway . Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway . Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p . Deletion of NPR1 did not affect the quantity, phosphorylation state, or localization of Ras2p . Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubiquitin suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway . Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate. Mol Cell Biol, 2001 Mar, 21(5), 1719 - 29 Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage; Anderson L et al.; 53BP1 is a human BRCT protein that was originally identified as a p53-interacting protein by the Saccharomyces cerevisiae two-hybrid screen . Although the carboxyl-terminal BRCT domain shows similarity to Crb2, a DNA damage checkpoint protein in fission yeast, there is no evidence so far that implicates 53BP1 in the checkpoint . We have identified a Xenopus homologue of 53BP1 (XL53BP1) . XL53BP1 is associated with chromatin and, in some cells, localized to a few large foci under normal conditions . Gamma-ray irradiation induces increased numbers of the nuclear foci in a dose-dependent manner . The damage-induced 53BP1 foci appear rapidly (in 30 min) after irradiation, and de novo protein synthesis is not required for this response . In human cells, 53BP1 foci colocalize with Mrel1 foci at later stages of the postirradiation period . XL53BP1 is hyperphosphorylated after X-ray irradiation, and inhibitors of ATM-related kinases delay the relocalization and reduce the phosphorylation of XL53BP1 in response to X-irradiation . In AT cells, which lack ATM kinase, the irradiation-induced responses of 53BP1 are similarly affected . These results suggest a role for 53BP1 in the DNA damage response and/or checkpoint control which may involve signaling of damage to p53. Mol Cell Biol, 2001 Mar, 21(5), 1515 - 30 Upf1p, Nmd2p, and Upf3p regulate the decapping and exonucleolytic degradation of both nonsense-containing mRNAs and wild-type mRNAs; He F et al.; In Saccharomyces cerevisiae, rapid degradation of nonsense-containing mRNAs requires the decapping enzyme Dcp1p, the 5'-to-3' exoribonuclease Xrn1p, and the three nonsense-mediated mRNA decay (NMD) factors, Upf1p, Nmd2p, and Upf3p . To identify specific functions for the NMD factors, we analyzed the mRNA decay phenotypes of yeast strains containing deletions of DCP1 or XRN1 and UPF1, NMD2, or UPF3 . Our results indicate that Upf1p, Nmd2p, and Upf3p regulate decapping and exonucleolytic degradation of nonsense-containing mRNAs . In addition, we show that these factors also regulate the same processes in the degradation of wild-type mRNAs . The participation of the NMD factors in general mRNA degradation suggests that they may regulate an aspect of translation termination common to all transcripts. J Virol, 2001 Apr, 75(7), 3207 - 19 Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a 5' proximal RNA2 signal; Chen J et al.; Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors . Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions . 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain . In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane . Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication . To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo . In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a . In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane . Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5'-proximal third of RNA2 . The RNA2 5' untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA . However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA . A 5'-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TPsiC stem-loop of host tRNAs . The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates . These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication. J Cell Biol, 2001 Mar 5, 152(5), 935 - 44 Rer1p, a retrieval receptor for endoplasmic reticulum membrane proteins, is dynamically localized to the Golgi apparatus by coatomer; Sato K et al.; Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins . We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal . A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER . Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole . The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing alpha and gamma subunits . These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p. Bioinformatics, 2001 Feb, 17(2), 196 - 7 MOSAIC: segmenting multiple aligned DNA sequences; Andre C et al.; MOSAIC is a set of tools for the segmentation of multiple aligned DNA sequences into homogeneous zones . The segmentation is based on the distribution of mutational events along the alignment . As an example, the analysis of one repeated sequence belonging to the subtelomeric regions of the yeast genome is presented . AVAILABILITY: Free access from ftp://ftp.biomath.jussieu.fr/pub/papers/MOSAIC J Mol Biol, 2001 Mar 9, 306(5), 957 - 68 MSH-MLH complexes formed at a DNA mismatch are disrupted by the PCNA sliding clamp; Bowers J et al.; In the yeast Saccharomyces cerevisiae, mismatch repair (MMR) is initiated by the binding of heterodimeric MutS homolog (MSH) complexes to mismatches that include single nucleotide and loop insertion/deletion mispairs . In in vitro experiments, the mismatch binding specificity of the MSH2-MSH6 heterodimer is eliminated if ATP is present . However, addition of the MutL homolog complex MLH1-PMS1 to binding reactions containing MSH2-MSH6, ATP, and mismatched substrate results in the formation of a stable ternary complex . The stability of this complex suggests that it represents an intermediate in MMR that is subsequently acted upon by other MMR factors . In support of this idea, we found that the replication processivity factor proliferating cell nuclear antigen (PCNA), which plays a critical role in MMR at step(s) prior to DNA resynthesis, disrupted preformed ternary complexes . These observations, in conjunction with experiments performed with streptavidin end-blocked mismatch substrates, suggested that PCNA interacts with an MSH-MLH complex formed on DNA mispairs. Dev Biol, 2001 Mar 15, 231(2), 410 - 9 Formation of the middle ear: recent progress on the developmental and molecular mechanisms; Mallo M; The middle ear allows animals to hear while moving in an aerial medium . It is composed of a cavity harbouring a chain of three ossicles that transmit vibrations produced by airborne sound in the tympanic membrane into the inner ear, where they are converted into neural impulses . The middle ear develops in the branchial arches, and this requires sequential interactions between the epithelia and the underlying mesenchyme . Gene-inactivation experiments have identified genes required for the formation of different middle ear components . Some encode for signalling molecules, including Endothelin1 and Fgf8, probable mediators of epithelial-mesenchymal interactions . Other genes, including Eya1, Prx1, Hoxa1, Hoxa2, Dlx1, Dlx2, Dlx5, and Gsc, are most likely involved in patterning and morphogenetic processes in the neural crest-derived mesenchyme . Mechanisms controlling formation of a functional tympanic membrane are also discussed . Basically, the tympanic ring, which serves as support for the tympanic membrane, directs invagination of the first pharyngeal cleft ectoderm to form the external acoustic meatus (EAM), which provides the outer layer of the membrane . Gsc and Prx1 are essential for tympanic ring development . While invaginating, the EAM controls skeletogenesis in the underlying mesenchyme to form the manubrium of the malleus, the link between the membrane and the middle ear ossicles . Nature, 2001 Feb 15, 409(6822), 839 - 41 A genomic perspective on membrane compartment organization; Bock JB et al.; Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families . To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes . Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm . All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits . In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies. Shock, 2001 Mar, 15(3), 165 - 70 Injury in the era of genomics; Cobb JP et al.; The traditional approach to the study of biology employs small-scale experimentation that results in the description of a molecular sequence of known function or relevance . In the era of the genome the reverse is true, as large-scale cloning and gene sequencing come first, followed by the use of computational methods to systematically determine gene function and regulation . The overarching goal of this new approach is to translate the knowledge learned from a systematic, global analysis of genomic data into a complete understanding of biology . For investigators who study shock, the specific goal is to increase understanding of the adaptive response to injury at the level of the entire genome . This review describes our initial experience using DNA microarrays to profile stress-induced changes in gene expression . We conclude that efforts to apply genomics to the study of injury are best coordinated by multi-disciplinary groups, because of the extensive expertise required. Biochem Cell Biol, 2001, 79(1), 83 - 91 A tale of two charges: distinct roles for an acidic and a basic amino acid in the structure and function of cytochrome c; Parrish JC et al.; Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria . As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history . Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp . There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined . Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electrostatically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular) . This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86 . Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90. RNA, 2001 Feb, 7(2), 275 - 84 Programmed +1 frameshifting stimulated by complementarity between a downstream mRNA sequence and an error-correcting region of rRNA; Li Z et al.; Like most retroviruses and retrotransposons, the retrotransposon Ty3 expresses its pol gene analog (POL3) as a translational fusion to the upstream gag analog (GAG3) . The Gag3-Pol3 fusion occurs by frameshifting during translation of the mRNA that encodes the two separate but overlapping ORFs . We showed previously that the shift occurs by out-of-frame binding of a normal aminoacyl-tRNA in the ribosomal A site caused by an aberrant codonoanticodon interaction in the P site . This event is unlike all previously described programmed translational frameshifts because it does not require tRNA slippage between cognate or near-cognate codons in the mRNA . A sequence of 15 nt distal to the frameshift site stimulates frameshifting 7.5-fold . Here we show that the Ty3 stimulator acts as an unstructured region to stimulate frameshifting . Its function depends on strict spacing from the site of frameshifting . Finally, the stimulator increases frameshifting dependent on sense codon-induced pausing, but has no effect on frameshifting dependent on pauses induced by nonsense codons . Complementarity between the stimulator and a portion of the accuracy center of the ribosome, Helix 18, implies that the stimulator may directly disrupt error correction by the ribosome. Yi Chuan Xue Bao, 2001, 28(2), 144 - 51 {Differential accumulation of the new high-affinity phosphate transporter candidated gene fragment in rice roots in response to phosphorus deficiency stress}; Yu FT et al.; Phosphate is a major constraint to crop production, and phosphate uptake in plant is mainly by high-affinity phosphate transporter under phosphate deficiency condition . Using RT-PCR, a 1,178 bp phosphate transporter gene fragment OjPT1 was cloned from roots of Jingxi17 (Oryza sativa L . ssp . japanica) supplied with no phosphate . The comparison of this sequence with ones in GenBank indicated that it shared about 70% similarity at amino acid level with other phosphate transporters in higher plants, such as Arabidopsis thaliana, potate, tamato, Medicago truncatula and Catharanthus roseus, and high similarity with phosphate transporters in Saccharomyces cerevisiae and Neurospora crassa . RT-PCR assay showed that the OjPT1 transcripts were induced under phosphate deficiency condition . This gene fragment OjPT1 has been deposited in GenBank (accession No . AF249619). Indian J Exp Biol, 2000 Jan, 38(1), 1 - 5 Ageing, gerontogenes, and hormesis; Rattan SI; Evolutionary theories of ageing and longevity argue against the existence of specific genes that cause ageing . However, genes whose altered activity influences ageing and longevity, may be termed gerontogenes . Several putative gerontogenes have been identified in various ageing systems, including the Drosophila, budding yeast, nematodes and cells in culture . Since ageing is characterized by a progressive failure of maintenance and repair, it is reasoned that genes involved in homeodynamic repair pathways are the most likely candidate gerontogenes . A promising approach for the identification of critical gerontogenic processes is hormesis-like positive effects of stress . Stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms. Eur J Biochem, 2001 Mar, 268(5), 1238 - 49 Phosphoinositide fatty acids regulate phosphatidylinositol 5-kinase, phospholipase C and protein kinase C activities; Carricaburu V et al.; PtdIns(4,5)P(2) generally results from phosphorylation of PtdIns(4)P by the phosphatidylinositol 5-kinase (PtdIns5-K) . Its hydrolysis by phospholipase C (PLC) yields inositol 1,4,5-trisphosphate and diacylglycerol, which stimulates protein kinase C (PKC) . We show that epithelial cells of the cockroach rectum contain three different inositol lipids: PtdIns(4,5)P(2), PtdIns(4)P, and PtdIns . They are composed of six major fatty acids: palmitic (16:0) stearic (18:0), oleic (18:1n--9), linoleic (18:2n--6), linolenic (18:3n--3), and arachidonic (20:4n-6) acids . The fatty acid preference of each of the above enzymes was evaluated by incorporating different fatty acids in pairs into membrane lipids . Incorporation of 16:0 plus 18:1n--9 provoked an increase in PtdIns(4,5)P2-PLC activity and a decrease in PtdIns5-K activity . In contrast, incorporation of 16:0 plus 18:3n--3 led to a potentiation of PtdIns5-K activity and a decrease in PtdIns(4,5)P(2)-PLC activity . Furthermore, PLC and PtdIns5-K acted preferentially on substrates containing 18:3n--3, and 18:3n--3-containing diacylglycerol specifically potentiated PKC activity . Thus, we propose that the fatty acids that make up the phosphoinositides function as intracellular modulators of the activity of certain enzymes. Curr Biol, 2001 Feb 6, 11(3), R109 - 11 Membrane transport: retromer to the rescue; Pfeffer SR; Genetic analysis in yeast has led to the discovery of a complex that retrieves proteins selectively from the prevacuolar compartment and transports them to the Golgi . Orthologs of these proteins in mammalian cells are likely to play a similar role but their cargoes are yet to be identified. Curr Biol, 2001 Jan 23, 11(2), R45 - 8 Meiotic recombination: Making and breaking go hand in hand; Baudat F et al.; Accurate segregation of homologous chromosomes at the first meiotic division requires the tight coordination of DNA replication, homologous recombination and chromosome organization . Recent studies suggest that the initiation of meiotic recombination is mechanistically coupled to premeiotic DNA replication. Curr Biol, 2001 Jan 23, 11(2), 99 - 104 enok encodes a Drosophila putative histone acetyltransferase required for mushroom body neuroblast proliferation; Scott EK et al.; Mushroom bodies in the Drosophila brain are centers for olfactory learning and memory . We have previously shown that the mushroom bodies comprise three types of neurons with distinct axonal projections . These three types of neurons are generated sequentially from common neuroblasts . We report here the identification of a gene that we have named enoki mushroom (enok), which when it is mutated gives rise to mushroom bodies with reduced axonal structures . enok encodes a putative histone acetyltransferase (HAT) of the MYST family, members of which have been implicated as important modulators of transcriptional activity . A single amino acid change in the zinc finger motif of the putative catalytic HAT domain gives the same phenotype as a null allele, and this finding indicates the importance of HAT activity to Enok's function . Further phenotypic analysis demonstrates that the mushroom body defect is due to an arrest in neuroblast proliferation rather than a failure of either cell fate switching or axon branching . Clonal analyses in the wing discs and the ovaries suggest that enok is essential for normal cell proliferation in some, but not all, tissues . Our results provide in vivo evidence for essential functions of a histone acetyltransferase in the construction of the Drosophila brain. Plant Cell Physiol, 2001 Feb, 42(2), 231 - 5 Novel family of sensor histidine kinase genes in Arabidopsis thaliana; Ueguchi C et al.; We identified three novel, highly homologous, sensor histidine kinases that possibly function in the plasma membrane of Arabidopsis thaliana, i.e . AHK2, 3 and 4 . While AHK2 and 3 are expressed in several organs, AHK4 is mainly expressed in roots . AHK3 suppresses a sensor histidine kinase mutant of yeast. Mol Biol Evol, 2001 Mar, 18(3), 330 - 43 Tracing the origin of the compensasome: evolutionary history of DEAH helicase and MYST acetyltransferase gene families; Sanjuan R et al.; Dosage compensation in Drosophila is mediated by a complex of proteins and RNAs called the "compensasome." Two of the genes that encode proteins of the complex, maleless (mle) and males-absent-on-the-first (mof), respectively, belong to the DEAH helicase and MYST acetyltransferase gene families . We performed comprehensive phylogenetic and structural analyses to determine the evolutionary histories of these two gene families and thus to better understand the origin of the compensasome . All of the members of the DEAH and MYST families of the completely sequenced Saccharomyces cerevisiae and Caenorhabditis elegans genomes, as well as those so far (June 2000) found in Drosophila melanogaster (for which the euchromatic part of the genome has also been fully sequenced) and Homo sapiens, were analyzed . We describe a total of 39 DEAH helicases in these four species . Almost all of them can be grouped in just three main branches . The first branch includes the yeast PRP2, PRP16, PRP22, and PRP43 splicing factors and their orthologs in animal species . Each PRP gene has a single ortholog in metazoans . The second branch includes just four genes, found in yeast (Ecm16) and Drosophila (kurz) and their orthologs in humans and Caenorhabditis . The third branch includes (1) a single yeast gene (YLR419w); (2) six Drosophila genes, including maleless and spindle-E/homeless; (3) four human genes, among them the ortholog of maleless, which encodes RNA helicase A; and (4) three C . elegans genes, including orthologs of maleless and spindle-E . Thus, this branch has largely expanded in metazoans . We also show that, for the whole DEAH family, only MLE and its metazoan orthologs have acquired new protein domains since the fungi/animals split . We found a total of 17 MYST family proteins in the four analyzed species . We determined putative orthologs of mof in both C . elegans and H . sapiens, and we show that the most likely ortholog in yeast is the Sas2 gene . Moreover, a paralog of mof exists in Drosophila . All of these results, together with those found for a third member of the compensasome, msl-3, suggest that this complex emerged after the fungi/animals split and that it may be present in mammalian species . Both gene duplication and the acquisition of new protein modules may have played important roles in the origin of the compensasome. Hum Mol Genet, 2001 Mar 15, 10(6), 635 - 43 Mutations in the regulatory domain of cystathionine beta synthase can functionally suppress patient-derived mutations in cis; Shan X et al.; Human cystathionine beta--synthase (CBS) is an S-adenosylmethionine-regulated enzyme that plays a key role in the metabolism of homocysteine . Mutations in CBS are known to cause homocystinuria, an inborn error in metabolism . We previously developed a yeast functional assay for CBS and used it to characterize mutations found in homocystinuric patients . We discovered that many patient-derived mutations are functionally suppressed by deletion of the C-terminal 142 amino acids, which contain a 53 amino acid motif known as the CBS domain . This domain is found in a wide variety of proteins of diverse biological function . Here we have used a genetic screen to identify missense mutations in the C-terminal region of CBS that can suppress the most common patient mutation, I278T . Seven suppressor mutations were identified, four of which map to the CBS domain . When combined in cis with another pathogenic mutation, V168M, six of seven of the suppressor mutations rescued the yeast phenotype . Enzyme activity analyses indicate that the suppressors restore activity from <2% to 17--64% of the wild-type levels . Analysis of the suppressor mutations in the absence of the pathogenic mutation shows that six of the seven suppressor alleles have lost enzymatic responsiveness to S-adenosylmethionine . Using homology modeling, we show that the suppressor mutations appear to map on one face of the CBS domain . Our results indicate that subtle changes to the C-terminus of CBS can restore activity to mutant proteins and provide a rationale for screening for compounds that can activate mutant CBS alleles. Genome Res, 2001 Mar, 11(3), 373 - 81 Gene duplication and the structure of eukaryotic genomes; Friedman R et al.; A simple method for understanding how gene duplication has contributed to genomic structure was applied to the complete genomes of Caenorhabditis elegans, Drosophila melanogaster, and yeast Saccharomyces cerevisiae . By this method, the genes belonging to gene families (the paranome) were identified, and the extent of sharing of two or more families between genomic windows was compared with that expected under a null model . The results showed significant evidence of duplication of genomic blocks in both C . elegans and yeast . In C . elegans, the five block duplications identified all occurred intra-chromosomally, and all but one occurred quite recently . In yeast, by contrast, 39 duplicated blocks were identified, and all but one of these was inter-chromosomal . Of these 39 blocks, 28 showed evidence of ancient duplication, possibly as a result of an ancient polyploidization event . By contrast, three blocks showed evidence of very recent duplication, while seven others showed a mixture of ancient and recent duplication events . Thus, duplication of genomic blocks has been an ongoing feature of yeast evolution over the past 200--300 million years. EMBO J, 2001 Mar 1, 20(5), 1173 - 83 Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13; Grandin N et al.; In Saccharomyces cerevisiae, Cdc13 has been proposed to mediate telomerase recruitment at telomere ends . Stn1, which associates with Cdc13 by the two-hybrid interaction, has been implicated in telomere maintenance . Ten1, a previously uncharacterized protein, was found to associate physically with both Stn1 and Cdc13 . A binding defect between Stn1-13 and Ten1 was responsible for the long telomere phenotype of stn1-13 mutant cells . Moreover, rescue of the cdc13-1 mutation by STN1 was much improved when TEN1 was simultaneously overexpressed . Several ten1 mutations were found to confer telomerase-dependent telomere lengthening . Other, temperature-sensitive, mutants of TEN1 arrested at G(2)/M via activation of the Rad9-dependent DNA damage checkpoint . These ten1 mutant cells were found to accumulate single-stranded DNA in telomeric regions of the chromosomes . We propose that Ten1 is required to regulate telomere length, as well as to prevent lethal damage to telomeric DNA. EMBO J, 2001 Mar 1, 20(5), 1134 - 43 A novel mRNA-decapping activity in HeLa cytoplasmic extracts is regulated by AU-rich elements; Gao M et al.; While decapping plays a major role in mRNA turnover in yeast, biochemical evidence for a similar activity in mammalian cells has been elusive . We have now identified a decapping activity in HeLa cytoplasmic extracts that releases (7me)GDP from capped transcripts . Decapping is activated in extracts by the addition of (7me)GpppG, which specifically sequesters cap-binding proteins such as eIF4E and the deadenylase DAN/PARN . Similar to in vivo observations, the presence of a poly(A) tail represses decapping of RNAs in vitro in a poly(A)-binding protein-dependent fashion . AU-rich elements (AREs), which act as regulators of mRNA stability in vivo, are potent stimulators of decapping in vitro . The stimulation of decapping by AREs requires sequence-specific ARE-binding proteins . These data suggest that cap recognition and decapping play key roles in mediating mRNA turnover in mammalian cells. EMBO J, 2001 Mar 1, 20(5), 1123 - 33 Regulation of the Sko1 transcriptional repressor by the Hog1 MAP kinase in response to osmotic stress; Proft M et al.; Exposure of yeast to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which is essential for the induction of gene expression required for cell survival upon osmotic stress . Several genes are regulated in response to osmotic stress by Sko1, a transcriptional repressor of the ATF/CREB family . We show by in vivo coprecipitation and phosphorylation studies that Sko1 and Hog1 interact and that Sko1 is phosphorylated upon osmotic stress in a Hog1-dependent manner . Hog1 phosphorylates Sko1 in vitro at multiple sites within the N-terminal region . Phosphorylation of Sko1 disrupts the Sko1-Ssn6-Tup1 repressor complex, and consistently, a mutant allele of Sko1, unphosphorylatable by Hog1, exhibits less derepression than the wild type . Interestingly, Sko1 repressor activity is further enhanced in strains with high protein kinase A (PKA) activity . PKA phosphorylates Sko1 near the bZIP domain and mutation of these sites eliminates modulation of Sko1 responses to high PKA activity . Thus, Sko1 transcriptional repression is controlled directly by the Hog1 MAPK in response to stress, and this effect is further modulated by an independent signaling mechanism through the PKA pathway. EMBO J, 2001 Mar 1, 20(5), 951 - 60 The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria; Wiedemann N et al.; The ADP/ATP carrier (AAC) is a major representative of mitochondrial preproteins lacking an N-terminal presequence . AAC contains targeting information in each of its three modules, which has led to a search for the dominant targeting region . An alternative, not yet tested model would be that several distinct targeting signals function simultaneously in import of the preprotein . We report that the three AAC modules cooperate in binding to the receptor Tom70 such that three Tom70 dimers are recruited to one preprotein . The modules are transferred to the import pore in a stepwise manner and cooperate again in the accumulation of AAC in the general import pore complex . AAC can cross the outer membrane with an internal segment first, i.e . in a loop formation . Each module of AAC is required for dimerization in the inner membrane . We propose a new concept for import of the hydrophobic carrier proteins into mitochondria where multiple signals cooperate in receptor recruitment, outer membrane translocation via loop formation and assembly in the inner membrane. EMBO J, 2001 Mar 1, 20(5), 941 - 50 The mitochondrial Hsp70-dependent import system actively unfolds preproteins and shortens the lag phase of translocation; Lim JH et al.; Unfolding is an essential process during translocation of preproteins into mitochondria; however, controversy exists as to whether mitochondria play an active role in unfolding . We have established an in vitro system with a kinetic saturation of the mitochondrial import machinery, yielding translocation rates comparable to in vivo import rates . Preproteins with short N-terminal segments in front of a folded domain show a characteristic delay of the onset of translocation (lag phase) although the maximal import rate is similar to that of longer preproteins . The lag phase is shortened by extending the N-terminal segment to improve the accessibility to matrix heat shock protein 70 and abolished by unfolding of the preprotein . A mutant mtHsp70 defective in binding to the inner membrane prolongs the lag phase and reduces the translocation activity . A direct comparison of the rate of spontaneous unfolding in solution with that during translocation demonstrates that unfolding by mitochondria is significantly faster, proving an active unfolding process . We conclude that access of mtHsp70 to N-terminal preprotein segments is critical for active unfolding and initiation of translocation. Antioxid Redox Signal, 2000 Fall, 2(3), 397 - 404 Tocopherol-binding proteins: their function and physiological significance; Stocker A et al.; The present review is a continuation of earlier essays on the uptake mechanisms and the biological function of vitamin E . There are eight naturally occurring homologues of vitamin E, which differ in their structure and in biological activity in vivo and in vitro . Various studies have suggested that after normal gastrointestinal absorption of dietary vitamin E specific mechanisms favor the preferential accumulation of one of its homologues, alpha-tocopherol, in the human body . This process is thought to be mediated in part by the alpha-tocopherol transfer protein (alpha-TTP) in the liver cytoplasm . The mechanism and pathway by which alpha-TTP specifically incorporates alpha-tocopherol into plasma lipoproteins is not yet fully understood . Because alpha-tocopherol is widely distributed in tissues in various concentrations but alpha-TTP resides only in liver, its role as intracellular carrier of alpha-tocopherol seems unlikely . However, recent data indicate that a system of alpha-tocopherol-binding proteins is involved in these processes that favor the localization of alpha-tocopherol at the sites where it is required . The current status of the evidence for the regulation of alpha-tocopherol levels and their impact on cellular signaling is discussed. Leuk Res, 2001 Mar, 25(3), 241 - 7 Phorbol ester responsiveness of the glutathione S-transferase P1 gene promoter involves an inducible c-jun binding in human K562 leukemia cells; Borde-Chiche P et al.; Overexpression of the glutathione S-transferase P1 (GSTP1) gene is related to drug resistance in human cancer cells . However, the mechanisms of the transcriptional activation of this gene remain unclear . In this study, we examined the molecular mechanisms underlying phorbol ester mediated gene regulation using human K562 leukemia cells as a model . Promoter deletion analyses revealed that the activator protein-1 (AP-1) transcription factor site was crucial for 12-O-tetradecanoyl phorbol 13-acetate (TPA)-mediated GSTP1 gene transcription . Electrophoretic mobility shift assays and transient transfection analysis demonstrated that both DNA binding and transactivation activities of AP-1 were induced by TPA . By supershift analysis, we identified transcription factors c-jun and fra-1 as well as NF-E2p45 as components of the induced binding complex . These results show for the first time that the phorbol ester TPA is involved in the molecular mechanism(s) mediating the activation of the GSTP1 promoter in a human leukemia model. Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2550 - 4 Interaction of a transcriptional repressor with the RNA polymerase II holoenzyme plays a crucial role in repression; Zaman Z et al.; The yeast transcriptional repressor Tup1, tethered to DNA, represses to strikingly different degrees transcription elicited by members of two classes of activators . Repression in both cases is virtually eliminated by mutation of either member of the cyclin-kinase pair Srb10/11 . In contrast, telomeric chromatin affects both classes of activators equally, and in neither case is that repression affected by mutation of Srb10/11 . In vitro, Tup1 interacts with RNA polymerase II holoenzyme bearing Srb10 as well as with the separated Srb10 . These and other findings indicate that at least one aspect of Tup1's action involves interaction with the RNA polymerase II holoenzyme. EMBO J, 2001 Jan 15, 20(1-2), 250 - 61 PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER; Maeda Y et al.; Glycosylphosphatidylinositol (GPI) acts as a membrane anchor of many cell surface proteins . Its structure and biosynthetic pathway are generally conserved among eukaryotic organisms, with a number of differences . In particular, mammalian and protozoan mannosyltransferases needed for addition of the first mannose (GPI-MT-I) have different substrate specificities and are targets of species- specific inhibitors of GPI biosynthesis . GPI-MT-I, however, has not been molecularly characterized . Characterization of GPI-MT-I would also help to clarify the topology of GPI biosynthesis . Here, we report a human cell line defective in GPI-MT-I and the gene responsible, PIG-M . PIG-M encodes a new type of mannosyltransferase of 423 amino acids, bearing multiple transmembrane domains . PIG-M has a functionally important DXD motif, a characteristic of many glycosyltransferases, within a domain facing the lumen of the endoplasmic reticulum (ER), indicating that transfer of the first mannose to GPI occurs on the lumenal side of the ER membrane. EMBO J, 2001 Jan 15, 20(1-2), 118 - 27 Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1; Holm M et al.; Arabidopsis COP1 is a photomorphogenesis repressor capable of directly interacting with the photomorphogenesis-promoting factor HY5 . This interaction between HY5 and COP1 results in targeted deg radation of HY5 by the 26S proteasome . Here we characterized the WD40 repeat domain-mediated interactions of COP1 with HY5 and two new proteins . Mutational analysis of those interactive partners revealed a conserved motif responsible for the interaction with the WD40 domain . This novel motif, with the core sequence V-P-E/D-&phi;-G (&phi; = hydrophobic residue) in conjunction with an upstream stretch of 4-5 negatively charged residues, interacts with a defined surface area of the ss-propeller assembly of the COP1 WD40 repeat domain through both hydrophobic and ionic interactions . Several residues in the COP1 WD40 domain that are critical for the interaction with this motif have been revealed . The fact that point mutations either in the COP1 WD40 domain or in the HY5 motif that abolish the interaction between COP1 and HY5 in yeast result in a dramatic reduction of HY5 degradation in transgenic plants validates the biological significance of this defined interaction. Fresenius J Anal Chem, 2000 Feb, 366(3), 303 - 6 Application of magdala red as a fluorescence probe in the determination of nucleic acids; Yang HH et al.; A fluorescence quenching method was developed for the rapid determination of DNA and RNA using magdala red as fluorescence probe . In weakly acidic medium, the fluorescence of magdala red (lambdaex/lambdaem = 540/555 nm) can be largely quenched by DNA or RNA . The calibration graphs are linear over the range 0.01-1.2 microg/mL for both calf thymus DNA (CT DNA) and salmon DNA (SM DNA), and 0.015-1.0 microg/mL for yeast RNA, respectively . The corresponding detection limits are 6.0 ng/mL for CT DNA, 7.0 ng/mL for SM DNA and 15.0 ng/mL for yeast RNA, respectively . CT DNA could be determined in the presence of 20% (w/w) yeast RNA, and the relative standard deviation of six replicate measurements is 3.18% for 400 ng/mL of CT DNA . Interference from coexisting substances in the determination of DNA was also examined . Real samples were determined with satisfactory results. Cancer Res, 2001 Feb 1, 61(3), 943 - 9 Phorbol esters modulate the Ras exchange factor RasGRP3; Lorenzo PS et al.; RasGRP represents the prototype of a new class of guanine nucleotide exchange factors that activate small GTPases . The guanyl nucleotide-releasing protein (GRP) family members contain catalytic domains related to CDC25, the Ras exchange factor of Saccharomyces cerevisiae . They also contain a motif resembling a pair of calcium-binding EF-hands and a C1 domain similar to the diacylglycerol interaction domain of protein kinase C . The sequence of KIAA0846, identified in a human brain cDNA library, encodes a member of the GRP family that we refer to as RasGRP3 . We show here that RasGRP3 bound phorbol esters with high affinity . This binding depended on anionic phospholipids, which is characteristic of phorbol ester binding to C1 domain proteins . In addition, phorbol esters also caused activation of the RasGRP3 exchange activity in intact cells, as determined by an increase in RasGTP and phosphorylation of the extracellular-regulated kinases . Finally, both phorbol 12-myristate 13-acetate and the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol induced redistribution of RasGRP3 to the plasma membrane and/or perinuclear area in HEK-293 cells, as demonstrated using a green fluorescent fusion protein . We conclude that RasGRP3 serves as a PKC-independent pathway to link the tumor-promoting phorbol esters with activation of Ras GTPases. Cancer Res, 2001 Feb 1, 61(3), 854 - 8 A mammalian two-hybrid system for adenomatous polyposis coli-mutated colon cancer therapeutics; Wakita K et al.; Colon cancer cells frequently lose expression of the tumor suppressor adenomatous polyposis coli (APC) . As result, beta-catenin accumulates and activates transcription of Tcf-responsive genes . Here we describe a novel mammalian two-hybrid system that selectively kills APC-mutated cells . This system consists of GAL4/beta-catenin, VP16/Tcf4, and a gene that is transcribed when GAL4 and VP16 associate . In APC-mutated human colon cancer cells, such as SW480, GAL4/beta-catenin accumulates, and in the presence of VP16/Tcf4, induces high levels of expression of the reporter gene . Expression of wild-type APC reduced GAL4/beta-catenin and intact beta-catenin levels and inhibited reporter gene expression . In colon cancer cells such as SW48 that have wild-type APC, GAL4/beta-catenin was degraded, and expression levels of the output gene were low . Replacement of the reporter gene with a suicide gene resulted in selective killing of SW480 cells . This system may be applicable for broader use of gene therapy by targeting diseases that involve protein degradation. Cancer Res, 2001 Feb 1, 61(3), 1095 - 9 Characterization of the major histocompatibility complex class I deficiencies in B16 melanoma cells; Seliger B et al.; The murine B16 melanoma system represents an important in vivo model for the evaluation of T cell-based immunization and vaccination strategies, although deficient MHC class I surface expression has been identified in these cells . We postulate here that the MHC class I-deficient phenotype of B16 melanoma cells is attributable to down-regulation or the loss of the expression and function of multiple components of the MHC class I antigen-processing pathway, including the peptide transporter associated with antigen processing, the proteasome subunits LMP2, LMP7, and LMP10, PA28alpha and -beta, and the chaperone tapasin . In contrast, calnexin, calreticulin, ER60, and protein disulfide isomerase expression are unaltered or only marginally suppressed in these cells . The level of down-regulation of the components of the antigen-processing pathway is either transcriptionally or posttranscriptionally controlled and could be corrected in all cases by IFN-y treatment, which also reconstituted MHC class I surface expression . Thus, B16 melanoma cells can be used as a model for the characterization of the mechanisms underlying the coordinated dysregulation of the antigen-processing components, which should provide new insights into the development of tumors and the factors controlling this process. Nat Struct Biol, 2001 Mar, 8(3), 258 - 64 Vam3p structure reveals conserved and divergent properties of syntaxins; Dulubova I et al.; Syntaxins and Sec1/munc18 proteins are central to intracellular membrane fusion . All syntaxins comprise a variable N-terminal region, a conserved SNARE motif that is critical for SNARE complex formation, and a transmembrane region . The N-terminal region of neuronal syntaxin 1A contains a three-helix domain that folds back onto the SNARE motif forming a 'closed' conformation; this conformation is required for munc18-1 binding . We have examined the generality of the structural properties of syntaxins by NMR analysis of Vam3p, a yeast syntaxin essential for vacuolar fusion . Surprisingly, Vam3p also has an N-terminal three-helical domain despite lacking apparent sequence homology with syntaxin 1A in this region . However, Vam3p does not form a closed conformation and its N-terminal domain is not required for binding to the Sec1/munc18 protein Vps33p, suggesting that critical distinctions exist in the mechanisms used by syntaxins to govern different types of membrane fusion. Yeast, 2001 Mar 15, 18(4), 323 - 4 YJL159w does encode Pir2/Hsp150; Moukadiri I et al.; In this paper we compare the sequence of the gene HSP150/PIR2, independently determined by two different groups, with that present in the yeast database as YJL159w, determined within the Yeast Sequencing Project . Although YJL159w is believed to encode Hsp150/Pir2, there are important differences between the amino acid sequence coded by this ORF and that of HSP150/PIR2 . To find out if this divergence is due to strain polymorphism or to a possible sequencing error, we have analysed the diverging zone of this ORF in three strains and have found it entirely consistent with the sequence reported as HSP150/PIR2, concluding that the divergence is probably due to a sequencing error in YJL159w . J Comp Neurol, 2001 Mar 19, 431(4), 481 - 91 Innervation of the ring gland of Drosophila melanogaster; Siegmund T et al.; In insects, peptidergic neurons of the central nervous system regulate the synthesis of the main developmental hormones . Neuropeptides involved in this neuroendocrine cascade have been identified in lepidopterans and dictyopterans . Since these organisms are not suitable for genetic research, we identified peptidergic brain neurons innervating the ring gland in Drosophila melanogaster . In larvae of Drosophila, ecdysteroids and juvenile hormones are produced by the ring gland, which is composed of the prothoracic gland, the corpus allatum, and the corpora cardiaca . Using the GAL4 enhancer trap system, we mapped those neurons of the central nervous system that innervate the ring gland . Eleven groups of neurosecretory neurons and their target tissues were identified . Five neurons of the lateral protocerebrum directly innervate the prothoracic gland or corpus allatum cells of the ring gland and are believed to regulate ecdysteroid and juvenile hormone titers . Axons of the circadian pacemaker neurons project onto dendritic fields of these five neurons . This connection might be the neuronal substrate of the circadian rhythms of molting and metamorphosis in Drosophila . Most of the neurons presented here have not been described before . The enhancer trap lines labeling them will be valuable tools for the analysis of neuronal as well as genetic regulation in insect development . Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 459 - 61 Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases; Blanchard H et al.; Forkhead-associated (FHA) domains are modular protein-protein interaction domains of approximately 130 amino acids present in numerous signalling proteins . FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules . FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized . Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 127.3, c = 386.3 A; diffraction data have been collected to 3.1 A resolution on a synchrotron source . Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 A resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 A. Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 393 - 400 Crystallographic phasing of myristoyl-CoA-protein N-myristoyltransferase using an iodinated analog of myristoyl-CoA; Futterer K et al.; Myristoyl-CoA-protein N-myristoyltransferase (Nmt; E.C . 2.1.3.97) catalyzes the covalent attachment of myristate to the N-terminal glycine amine of many eukaryotic and viral proteins . The molecular structure of the ternary complex of Saccharomyces cerevisiae Nmt1p with a bound non-hydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, and a competitive peptidomimetic inhibitor, SC-58272, was solved to 2.9 A resolution by X-ray crystallography . The structure determination utilized diffraction data from an iodinated ternary complex in which a newly designed and synthesized compound, S-(13-iodo-2-oxotridecyl)-CoA, was substituted for S-(2-oxopentadecyl)-CoA . Replacing the two terminal fatty acid C atoms of myristate by iodine produced, under the same crystallization conditions, heavy-atom-derivatized crystals of defined site occupancy that were isomorphous to the native complex . This approach for obtaining experimental phase information can be extended to other crystal structures of protein-fatty acyl complexes . The synthesis of S-(13-iodo-2-oxotridecyl)-CoA and the phasing procedure are described. Gene, 2001 Jan 24, 263(1-2), 31 - 8 Characterization of a MEN1 ortholog from Drosophila melanogaster; Guru SC et al.; Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized by tumors of the parathyroid, entero-pancreatic neuroendocrine and pituitary tissues and caused by inactivating mutations in the MEN1 gene . Menin, the 610-amino acid nuclear protein encoded by MEN1, binds to the transcription factor JunD and can repress JunD-induced transcription . We report here the identification of a MEN1 ortholog in Drosophila melanogaster, Menin1, that encodes a 763 amino acid protein sharing 46% identity with human menin . Additionally, 69% of the missense mutations and in-frame deletions reported in MEN1 patients appear in amino acid residues that are identical in the Drosophila and human protein, suggesting the importance of the conserved regions . Drosophila Menin1 gene transcripts use alternative polyadenylation sites resulting in 4.3 and 5-kb messages . The 4.3-kb transcript appears to be largely maternal, while the 5-kb transcript appears mainly zygotic . The binding of Drosophila menin to human JunD or Drosophila Jun could not be demonstrated by the yeast two-hybrid analysis . The identification of the MEN1 ortholog from Drosophila melanogaster will provide an opportunity to utilize Drosophila genetics to enhance our understanding of the function of human menin. FEBS Lett, 2001 Feb 16, 490(3), 179 - 89 Regulation of the G1 to S transition by the ubiquitin pathway; DeSalle LM et al.; This year the most prestigious prize in medical sciences, the Lasker Award, has been presented to the three scientists who discovered the ubiquitin pathway: Aaron Ciechanover, Avram Hershko, and Alexander Varshavsky {Nature Med . 6 (2000) 1073-1081} . During a time when the scientific community was focused on understanding how proteins were synthesized, they intently pursued the novel idea that cells were programmed to selectively destroy proteins . Their work led to the identification of an elaborate system of protein degradation targeting a myriad of cellular substrates . A small protein called ubiquitin is at the center of this process . Although the ubiquitin pathway was first described in the early 1980s, it has only more recently advanced to the forefront of basic research as a significant regulatory network within the cell . The field continues to grow as new ubiquitination enzymes and novel functions of this system are identified . Scientists are focused on elucidating the mechanisms by which cells deploy the ubiquitin pathway to control levels of selected proteins, such as cell cycle regulatory proteins, transcription factors and signaling molecules . Accelerated or decelerated rates of degradation of particular substrates participate in the genesis of many human diseases . Thus, understanding the mechanisms that confer specificity to the ubiquitin system will allow the development of novel therapeutic approaches to target aberrations in this pathway underlying tumorigenesis and other human pathologies. Insect Biochem Mol Biol, 2001 Mar 15, 31(4-5), 401 - 5 Thiafatty acids as tracers to investigate biosynthetic pathways of lepidopteran sex pheromones; Pinilla A et al.; In order to investigate the potential utility of thiafatty acids as tracers for biosynthetic studies of moth sex pheromones, a series of thiatetradecanoic acids, namely 8-, 9-, 10-, 11-, 12- and 13-thiatetradecanoic, were prepared and their metabolism was investigated in pheromone glands of Spodoptera littoralis . Analysis by gas chromatography coupled to mass spectrometry of extracts from pheromone glands treated with the above acids showed that only 8-thiatetradecanoic acid and 13-thiatetradecanoic acid were metabolized by desaturation and were incorporated into the sex pheromone biosynthetic pathway . 13-Thiatetradecanoic acid was converted into (E)- and (Z)-13-thiatetradec-11-enoic acids, (Z,E)-13-thiatetradeca-9,11-dienoic acid, 11-thiadodecanoic acid, (E)- and (Z)-11-thiadodec-9-enoic acids and 15-thiahexadecanoic acid . 8-Thiatetradecanoic acid gave rise to two monoenoic thiafatty acids and two dienoic thiafatty acids, which were assigned to (Z)- and (E)-8-thiatetradec-11-enoic acids, (Z,E)-8-thiatetradeca-9,11-dienoic acid and (E,E)-8-thiatetradeca-10,12-dienoic acid . The other thiafatty acids tested, 9-, 10-, 11- and 12-thiatetradecanoic acids, were not metabolized by desaturation, although the corresponding products of beta-oxidation and chain elongation were detected . The occurrence of sulfoxides was not detected in this case, in disagreement with results on the metabolism of some thiaacids previously reported by other authors in yeast, Saccharomyces cerevisiae. J Virol, 2001 Mar, 75(6), 2627 - 33 Antiviral response in cells containing Stat1 with heterologous transactivation domains; Shen Y et al.; The STATs (signal transducers and activators of transcription), latent cytoplasmic transcription factors, are activated by binding of extracellular polypeptides to cell surface receptors . Dimerization, accumulation in the nucleus, and transcriptional inductions of specific genes then occur . The COOH terminus of the STATs acts as a transcriptional activation domain (TAD) . Stat1, one of seven mammalian STAT genes, forms a homodimer after activation by gamma interferon and induces transcription of a number of genes . These induced genes in turn produce the antiviral state . In the present experiments we used a Stat1-deficient cell line complemented with Stat1 or various fusion constructs in which the wild-type Stat1 TAD was replaced by other TADs to test the possibility that a specific activating domain was necessary for the induction of the antiviral response . We found that a wide variety of TADs with different activation potential appended to the Stat1 COOH terminus could substitute for the wild-type protein in inducing the antiviral state. Blood, 2001 Mar 1, 97(5), 1266 - 73 Initiation of polyoma virus origin-dependent DNA replication through STAT5 activation by human granulocyte-macrophage colony-stimulating factor; Watanabe S et al.; Several lines of evidence indicate that transcriptional activation is coupled with DNA replication initiation, but the nature of initiation of DNA replication in mammalian cells is unclear . Polyoma virus replicon is an excellent system to analyze the initiation of DNA replication in murine cells because its replication requires an enhancer, and all components of replication machinery, except for DNA helicase large T antigen, are supplied by host cells . This system was used to examine the role of signal transducer and activator of transcription (STAT5) in replication initiation of polyoma replicon in the mouse lymphoid cell line BA/F3 . The plasmid with tandem repeats of consensus STAT5 binding sites followed by polyoma replication origin was replicated by stimulation with human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in the presence of polyoma large T antigen in BA/F3 cells . Mutation analysis of the hGM-CSF receptor beta subunit revealed that only the box1 region is essential, and the C-terminal tyrosine residues are dispensable for the activity . Addition of the tyrosine kinase inhibitor genistein suppressed this replication without affecting transcriptional activation of STAT5 . Because deletion analysis of STAT5 indicates the importance of the C-terminal transcriptional activation domain of STAT5 for the initiation of replication, the role of this region in the activation of replication was examined with a GAL4-STAT5 fusion protein . GAL4-STAT5 activated replication of the plasmid containing tandem repeats of GAL4 binding sites and polyoma replication origin in BA/F3 cells . Mutation analysis of GAL4-STAT5 indicated that multiple serine residues coordinately have a role in activating replication . This is the first direct evidence indicating the potential involvement of STAT5 in replication. Bioinformatics, 2001 Jan, 17(1), 44 - 57 Functional and structural genomics using PEDANT; Frishman D et al.; MOTIVATION: Enormous demand for fast and accurate analysis of biological sequences is fuelled by the pace of genome analysis efforts . There is also an acute need in reliable up-to-date genomic databases integrating both functional and structural information . Here we describe the current status of the PEDANT software system for high-throughput analysis of large biological sequence sets and the genome analysis server associated with it . RESULTS: The principal features of PEDANT are: (i) completely automatic processing of data using a wide range of bioinformatics methods, (ii) manual refinement of annotation, (iii) automatic and manual assignment of gene products to a number of functional and structural categories, (iv) extensive hyperlinked protein reports, and (v) advanced DNA and protein viewers . The system is easily extensible and allows to include custom methods, databases, and categories with minimal or no programming effort . PEDANT is actively used as a collaborative environment to support several on-going genome sequencing projects . The main purpose of the PEDANT genome database is to quickly disseminate well-organized information on completely sequenced and unfinished genomes . It currently includes 80 genomic sequences and in many cases serves as the only source of exhaustive information on a given genome . The database also acts as a vehicle for a number of research projects in bioinformatics . Using SQL queries, it is possible to correlate a large variety of pre-computed properties of gene products encoded in complete genomes with each other and compare them with data sets of special scientific interest . In particular, the availability of structural predictions for over 300 000 genomic proteins makes PEDANT the most extensive structural genomics resource available on the web. J Autoimmun, 2001 Feb, 16(1), 59 - 69 Monoclonal antibodies derived from BALB/c mice immunized with apoptotic Jurkat T cells recognize known autoantigens; Gensler TJ et al.; It has been postulated that post-translational modifications and relocalization of proteins during apoptosis may lead to presentation of these molecules to the immune system in such a way that normal mechanisms of tolerance are bypassed . In the present study, Jurkat cells were induced to undergo apoptosis by treatment with the chemotherapeutic agent Ara-C . BALB/c mice were then immunized with the apoptotic cells and hybridomas were generated . Using an indirect immunofluorescence assay, the monoclonal antibodies produced were screened by flow cytometry for those monoclonal antibodies demonstrating reactivity with permeabilized apoptotic Jurkat cells but not with non-permeabilized normal or apoptotic Jurkat cells . Of 281 monoclonal antibodies, 20 monoclonal antibodies with these properties were selected for further analysis . Using 32P- or 35S-metabolically labelled Jurkat cells, these selected monoclonal antibodies were screened for their ability to recognize autoantigens by immunoprecipitation and Western blotting . Well characterized autoimmune sera were then used to confirm the identity of autoantigens by immunoblotting . We demonstrate that immunization of normal mice with apoptotic Jurkat cells results in the formation of antibodies targeting multiple autoantigens or autoantigen complexes, including Ku, rRNPs, snRNPs and vimentin . These findings are consistent with the hypothesis that apoptosis can contribute to the development of autoimmunity. Biol Pharm Bull, 2001 Feb, 24(2), 144 - 50 Identification of zinc finger proteins bound to a silencer region in the rat glutathione transferase P gene; Tanabe A et al.; The rat glutathione transferase P (GST-P) gene is strongly induced during chemical hepatocarcinogenesis, whereas mRNA of this gene is rarely expressed in normal rat liver . We previously identified a silencer region in the promoter of this gene . This silencer has several DNA binding sites and at least three proteins (Silencer factor A, -B, and -C (SF-A, SF-B, and SF-C)) bind to these sites . We previously cloned and characterized the Nuclear Factor 1 (NF1) family and the CCAAT/enhancer-binding protein (C/EBP) family as SF-A and SF-B, respectively . However, SF-C which binds to GST-P silencer 2 (GPS2) remains to be cloned . By screening using yeast one-hybrid system, several zinc finger proteins were identified as a candidate of SF-C . The gel-mobility shift analyses showed that BTEB2, EZF, LKLF, TFIIIA, TIEG1, and novel zinc finger protein MZFP bound to GPS2 with different affinities . Several proteins of these are known to be transcriptional activators or repressors, suggesting that zinc finger proteins bind to GPS2 and regulate GST-P expression in the rat liver. Arzneimittelforschung, 2001 Jan, 51(1), 72 - 5 Antifungal activity of some mono, bis and quaternary Mannich bases derived from acetophenone; Gul HI et al.; The development of resistance to current antifungal therapeutics drives search for new effective agents . Some Mannich bases have antifungal activity, but no information is available regarding the antifungal activity of acetophenone derived Mannich bases . Mono Mannich bases of acetophenone 1-3 were synthesized and converted into their corresponding bis derivatives, 5-7 . Representative quaternary derivatives 4 and 8 were also synthesized . Antifungal activities of the compounds were evaluated using some yeasts and dermatophytes in vitro . Mono Mannich base 3 and quaternary compounds 4 and 8 were found to be 2-16 times more potent than the reference compound amphotericin B against dermatophytes: Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum canis . Compounds 4 and 8 were also found to be 2 times more effective compared with amphotericin B against the yeast Saccharomyces cerevisiae . Quaternization procedure improved the biological activity dramatically, whereas conversion of mono Mannich bases to corresponding bis derivatives generally did not affect antifungal activity . Our results suggest that acetophenone derived mono Mannich base 3 and quaternary derivatives 4 and 8 may serve as leading compounds for further studies to develop new antifungal agents with their highly potent antifungal activity. RNA, 2001 Jan, 7(1), 94 - 105 The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop; Shukla GC et al.; The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome . U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants . To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay . Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo . In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing . Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity . However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function . These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop. RNA, 2001 Jan, 7(1), 5 - 15 Evidence that phosphorylation of human Upfl protein varies with intracellular location and is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway; Pal M et al.; Human Upf1 protein (p), a group 1 RNA helicase, has recently been shown to function in nonsense-mediated mRNA decay (NMD) in mammalian cells . Here, we demonstrate that the estimated 3 x 10(6) copies of hUpf1 p per exponentially growing HeLa cell are essentially equally distributed among polysomal, subpolysomal, and ribosome-free fractions . We also demonstrate that hUpf1p binds RNA and is a phosphoprotein harboring phosphoserine and phosphothreonine . hUpf1p is phosphorylated to the highest extent when polysome-associated and to the lowest extent when ribosome free . We find that serum-induced phosphorylation of hUpf1p is inhibited by wortmannin at a concentration that selectively inhibits PI 3-kinase related kinases and, to a lesser extent, by rapamycin . These and other data suggest that phosphorylation is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway . Comparisons are made of hUpf1p to Upf1p and SMG-2, which are the orthologs to hUpf1p in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively. RNA, 2001 Jan, 7(1), 29 - 43 A ribozyme selected from variants of U6 snRNA promotes 2',5'-branch formation; Tuschl T et al.; In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated . One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing . The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine . It differs in that the leaving group is diphosphate rather than a 5' exon . The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation. Antioxid Redox Signal, 2000 Winter, 2(4), 801 - 10 The mitochondrial thioredoxin system; Miranda-Vizuete A et al.; Eukaryotic organisms from yeast to human possess a mitochondrial thioredoxin system composed of thioredoxin and thioredoxin reductase, similar to the cytosolic thioredoxin system that exists in the same cells . Yeast and mammalian mitochondrial thioredoxins are monomers of approximately 12 kDa and contain the typical conserved active site WCGPC . However, there are important differences between yeast and mammalian mitochondrial thioredoxin reductases that resemble the differences between their cytosolic counterparts . Mammalian mitochondrial thioredoxin reductase is a selenoprotein that forms a homodimer of 55 kDa/subunit; while yeast mitochondrial thioredoxin reductase is a homodimer of 37 kDa/subunit and does not contain selenocysteine . A function of the mitochondrial thioredoxin system is as electron donor for a mitochondrial peroxiredoxin, an enzyme that detoxifies the hydrogen peroxide generated by the mitochondrial metabolism . Experiments with yeast mutants lacking both the mitochondrial thioredoxin system as well as the mitochondrial peroxiredoxin system suggest an important role for mitochondrial thioredoxin, thioredoxin reductase, and peroxiredoxin in the protection against oxidative stress. J Environ Pathol Toxicol Oncol, 2000, 19(4), 401 - 13 Antimutagenesis studies of magnesium and calcium salts; Bronzetti G et al.; Magnesium is a microelement that is essential for biological functions and particularly for cellular metabolism . It has a central role in protein, lipid, carbohydrate, and nucleic acid synthesis, and it is important for muscular physiology and nerve excitability . Magnesium has an important role in the stability of biological membranes, it controls immune phenomena, and it activates over 300 enzymes . However, the mechanism of action of magnesium salts has not been well investigated and, in particular, its antimutagenesis properties and its effects in the detoxification of free radicals need further study . We investigated the effect of magnesium chloride, sulphate, carbonate, and oxide on the yeast Saccharomyces cerevisiae D7 strain, to evaluate their ability to protect against genotoxic damage . We found that magnesium salts induced antimutagenic effects in the cells harvested in the logarithmic phase by decreasing the induction of hydrogen peroxide . This, however, did not occur in the stationary phase . We also studied calcium salts of the type corresponding to those of magnesium and their protective role against the oxidative damage of free radicals and enzymatic activities, such as catalase, glutathione peroxidase, and superoxide dismutase, which are involved in antioxidative defenses. Cell Mol Life Sci, 1999 Oct 15, 56(3-4), 233 - 42 Myosin-V: head to tail; Provance DW et al.; The myosin-V family is the most extensively studied of the unconventional myosin families . Most organisms examined have at least one member of the myosin-V family: many have multiple members . The wide range of species in which myosin-V has been identified suggests that myosin-V is a fundamental component of organelle transport in all higher eukaryotes . Possible cargoes for myosin-V range from melanosomes and synaptic vesicles in mammals to vacuoles and messenger RNA in yeast . In this review, we discuss the current state of research on the cellular function of myosin-V as described by the actions of the head, neck and tail domains. Cell Mol Life Sci, 1999 Dec, 56(11-12), 894 - 907 Cathepsin A/protective protein: an unusual lysosomal multifunctional protein; Hiraiwa M; Cathepsin A/protective protein {3.4.16.5}, carboxypeptidase A, is a lysosomal serine protease with structural homology to yeast (Saccharomyces cerevisiae) carboxypeptidase Y . Cathepsin A is a member of the alpha/beta hydrolase fold family and has been suggested to share a common ancestral relationship with other alpha/beta hydrolase fold enzymes, such as cholinesterases . Several lines of evidence indicate that cathepsin A is a multicatalytic enzyme with deamidase and esterase in addition to carboxypeptidase activities . Cathepsin A was recently identified in human platelets as deamidase . In vitro, it hydrolyzes a variety of bioactive peptide hormones including tachykinins, suggesting that extralysosomal cathepsin A plays a role in regulation of bioactive peptide functions . Recent reports emphasize the lysosomal protective function of cathepsin A rather than its protease function . The protective function of cathepsin A is distinct from its catalytic function . Human lysosomal beta-galactosidase and neuraminidase exist as a high molecular weight enzyme complex, in which there is a 54-kDa glycoprotein termed 'lysosomal protective protein' . Based on cell culture studies, protective protein was found to protect both beta-galactosidase and neuraminidase from intralysosomal proteolysis by forming a multienzyme complex and was shown to be deficient in patients with galactosialidosis, a combined deficiency of beta-galactosidase and neuraminidase . Molecular cloning and gene expression studies have disclosed that protective protein is cathepsin A . The cathepsin A precursor has the potential to restore both beta-galactosidase and neuraminidase activities in fibroblasts from patients with galactosialidosis . Cathepsin A knockout mice showed a phenotype similar to human galactosialidosis and the deficient phenotype found in the mutant mice was corrected by transplanting erythroid precursor cells overexpressing cathepsin A . Collectively, these findings demonstrate the significance of cathepsin A as a key molecule in the onset of galactosialidosis and also highlight the therapeutic potential of the cathepsin A precursor for patients with galactosialidosis. Methods Enzymol, 2001, 329, 431 - 8 Purification and properties of rat liver Sec23-Sec24 complex; Weissman JT et al.; We have demonstrated a protocol for purifying functional Sec23-24 complex from rat liver cytosol . Because the rat liver Sec23-24 complex is highly susceptible to proteolysis, we have noted several modifications which have allowed us to overcome the problem of degradation . If care is taken to prevent proteolysis, this procedure typically yields 2 mg of functional complex . The Sec23-24 complex can then be used to study Sar1 GTP hydrolysis in the Sec23 GTPase activation assay . Additionally, Sec23-24 can reconstitute ER vesicle formation in the presence of Sar1 and Sec13-31, allowing for the identification of novel proteins or compounds that affect cargo export. Protein Sci, 2000 Dec, 9(12), 2470 - 6 Thirty-plus functional families from a single motif; Yu L et al.; It is now possible to identify over 30 functional subfamilies among the WD-repeat-containing proteins found in the completed genomes . The majority of these subfamilies have at least one member for which experimental data allow assignment to a cellular pathway or process . Half of the 63 WD-repeat-containing proteins in Saccharomyces cerevisiae, half of the 70 in Caenorhabditis elegans, and a third of the 100 plus predicted in Drosophila can be assigned to 23 of these functional subfamilies . Perhaps indicative of the future, 33 WD-repeat-containing proteins from the partial genome of Arabidopsis thaliana can now be assigned to 18 of these subfamilies . These assignments have been made possible by combining traditional sequence similarity with an implied common beta propeller structural context to obtain measures of protein-protein surface similarity . The beta propeller structural context is represented in the form of a Hidden Markov Model . The procedure is completely automated. Protein Sci, 2000 Dec, 9(12), 2457 - 69 The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis; Rath A et al.; We have characterized the thermodynamic stability of the SH3 domain from the Saccharomyces cerevisiae Abp1p protein and found it to be relatively low compared to most other SH3 domains, with a Tm of 60 degrees C and a deltaGu of 3.08 kcal/mol . Analysis of a large alignment of SH3 domains led to the identification of atypical residues at eight positions in the wild-type Abp1p SH3 domain sequence that were subsequently replaced by the residue seen most frequently at that position in the alignment . Three of the eight mutants constructed in this way displayed increases in Tm ranging from 8 to 15 degrees C with concomitant increases in deltaGu of up to 1.4 kcal/mol . The effects of these substitutions on folding thermodynamics and kinetics were entirely additive, and a mutant containing all three was dramatically stabilized with a Tm greater than 90 degrees C and a deltaGu more than double that of the wild-type domain . The folding rate of this hyperstable mutant was 10-fold faster than wild-type, while its unfolding rate was fivefold slower . All of the stabilized mutants were still able to bind a target peptide with wild-type affinity . We have analyzed the stabilizing amino acid substitutions isolated in this study and several other similar sequence alignment based studies . In approximately 25% of cases, increased stability can be explained by enhanced propensity of the substituted residue for the local backbone conformation at the mutagenized site. Philos Trans R Soc Lond B Biol Sci, 2001 Jan 29, 356(1405), 67 - 72 Evolution and the molecular basis of somatic hypermutation of antigen receptor genes; Diaz M et al.; Somatic hypermutation of immunoglobulin genes occurs in many vertebrates including sharks, frogs, camels, humans and mice . Similarities among species reveal a common mechanism and these include the AGC/T sequence hot spot, preponderance of base substitutions, a bias towards transitions and strand bias . There are some differences among species, however, that may unveil layers of the mechanism . These include a G:C bias in frog and shark IgM but not in nurse shark antigen receptor (NAR), a high frequency of doublets in NAR hypermutation, and the co-occurrence of somatic hypermutation with gene conversion in some species . Here we argue that some of the similarities and differences among species are best explained by error-prone DNA synthesis by the translesion synthesis DNA polymerase zeta (Pol zeta) and, as suggested by others, induction of DNA synthesis by DNA breaks in antigen receptor variable genes . Finally, targeting of the variable genes is probably obtained via transcription-related elements, and it is the targeting phase of somatic hypermutation that is the most likely to reveal molecules unique to adaptive immunity. Philos Trans R Soc Lond B Biol Sci, 2001 Jan 29, 356(1405), 41 - 6 Mutagenesis in eukaryotes dependent on DNA polymerase zeta and Rev1p; Lawrence CW et al.; DNA polymerase zeta (Pol zeta) and Rev1p carry out translesion replication in budding yeast, Saccharomyces cerevisiae, and are jointly responsible for almost all base pair substitution and frameshift mutations induced by DNA damage in this organism . In addition, Pol zeta is responsible for the majority of spontaneous mutations in yeast and has been proposed as the enzyme responsible for somatic hypermutability . Pol zeta, a non-processive enzyme that lacks a 3' to 5' exonuclease proofreading activity, is composed of Rev3p, the catalytic subunit, and a second subunit encoded by REV7 . In keeping with its role, extension by Pol zeta is relatively tolerant of abnormal DNA structure at the primer terminus and is much more capable of extension from terminal mismatches than yeast DNA polymerase alpha (Pol alpha) . Rev1p is a bifunctional enzyme that possesses a deoxycytidyl transferase activity that incorporates deoxycytidyl opposite abasic sites in the template and a second, at present poorly defined, activity that is required for the bypass of a variety of lesions as well as abasic sites . Human homologues of the yeast REV1 and REV3 have been identified and, based on the phenotype of cells producing antisense RNA to one or other of these genes, their products appear also to be employed in translation replication and spontaneous mutagenesis . We suggest that Pol zeta is best regarded as a replication enzyme, albeit one that is used only intermittently, that promotes extension at forks the progress of which is blocked for any reason, whether the presence of an unedited terminal mismatch or unrepaired DNA lesion. Arch Androl, 2001 Jan-Feb, 46(1), 29 - 35 Evidence for the binding of a human sperm component with diaphanous protein; Zhang SM et al.; The YWK-II component of human sperm membrane is related to the betaA4-amyloid precursor protein (APP) of Alzheimer's disease . A yeast 2-hybrid system was used to screen a mouse testis cDNA expression library for potential ligands capable of interacting with the extracellular domain of the YWK-II component . One of the bound proteins was identified as hDIA1, which has 96% identity with p140mDia . These proteins are members of the formin homology family and participate in cytokinesis and organization of the actin cytoskeleton . By interacting with these diaphanous proteins, the YWK-II component may be involved in germ cell differentiation and in the structural formation of the acrosome. Nature, 2001 Jan 18, 409(6818), 346 - 9 Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake; Curie C et al.; Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality . Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups . The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron . The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface . The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter . Here we show that ys1 is a membrane protein that mediates iron uptake . Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media . Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots . Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants. Plant Mol Biol, 2000 Nov, 44(5), 687 - 97 Hexose transporters of tomato: molecular cloning, expression analysis and functional characterization; Gear ML et al.; A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries . Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members . The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa . The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers . The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily . Expression of the three genes in different organs of tomato was investigated by quantitative PCR . LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips . LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers . LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae . LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H+/hexose symporter . The Km of the symporter for glucose is 45 microM. Faraday Discuss, 2000, (116), 119 - 34; discussion 171-90 Biomaterial engineered electrodes for bioelectronics; Pardo-Yissar V et al.; A series of single-cysteine-containing cytochrome c, Cyt c, heme proteins including the wild-type Cyt c (from Saccharomyces cerevisiae) and the mutants (V33C, Q21C, R18C, G1C, K9C and K4C) exhibit direct electrical contact with Au-electrodes upon covalent attachment to a maleimide monolayer associated with the electrode . With the G1C-Cyt c mutant, which includes the cysteine residue in the polypeptide chain at position 1, the potential-induced switchable control of the interfacial electron transfer was observed . This heme protein includes a positively charged protein periphery that surrounds the attachment site and faces the electrode surface . Biasing of the electrode at a negative potential (-0.3 V vs . SCE) attracts the reduced Fe(II)-Cyt c heme protein to the electrode surface . Upon the application of a double-potential-step chronoamperometric signal onto the electrode, where the electrode potential is switched to +0.3 V and back to -0.3 V, the kinetics of the transient cathodic current, corresponding to the re-reduction of the Fe(III)-Cyt c, is controlled by the time interval between the oxidative and reductive potential steps . While a short time interval results in a rapid interfacial electron-transfer, ket1 = 20 s-1, long time intervals lead to a slow interfacial electron transfer to the Fe(III)-Cyt c, ket2 = 1.5 s-1 . The fast interfacial electron-transfer rate-constant is attributed to the reduction of the surface-attracted Fe(III)-Cyt c . The slow interfacial electron-transfer rate constant is attributed to the electrostatic repulsion of the positively charged Cyt c from the electrode surface, resulting in long-range electron transfer exhibiting a lower rate constant . At intermediate time intervals between the oxidative and reductive steps, two populations of Cyt c, consisting of surface-attracted and surface-repelled heme proteins, are observed . Crosslinking of a layered affinity complex between the Cyt c and cytochrome oxidase, COx, on an Au-electrode yields an electrically-contacted, integrated, electrode for the four-electron reduction of O2 to water . Kinetic analysis reveals that the rate-limiting step in the bioelectrocatalytic reduction of O2 by the integrated Cyt c/COx electrode is the primary electron transfer from the electrode support to the Cyt c units. Genes Immun, 2000, 1(4), 237 - 50 Ku autoantigen (DNA helicase) is required for interleukins-13/-4-induction of 15-lipoxygenase-1 gene expression in human epithelial cells; Kelavkar UP et al.; As reported previously in human monocytes, a human lung epithelial cell line, A549, showed de novo induction of 15-Lipoxygenase-1 (15-LO-1) in response to interleukins-13 (IL-13) and -4 (IL-4) . In this cell line, 15-LO-1 expression, by RT-PCR and western blotting, was observed following 6 and 24 h of exposure to human IL-13 (ED50 5 ng/ml) and IL-4 (ED50 0.2 ng/ml) . We have previously shown that no cis-acting regulatory elements exist within the 15-LO-1 promoter region . To define IL-13 and IL-4 responsive trans-acting elements, we identified a region (DP2: -353 to -304 bp site) within the 15-LO-1 promoter (by footprinting experiments) to which IL-13-responsive elements (or factors) bind specifically (Kelavkar et al, 1998, Mol Biol Rep 25, 173-182) . To further delineate this region, we constructed (by site-directed mutagenesis) several deletion mutants in the 'LOPB5' region containing the 29 bp within the -353 to -304 bp of the DP2 core element . These were: DP3 (site totally deleted), DP4 (5 bp deleted at the center of the site), DP5 (8 bp at the 5'-end of the site) and DP6 (13 bp at the 3'-end of the site) . Cotransfection of these deletion constructs (driving luciferase reporter genes) was associated with 90% (DP4, DP5 and DP6) or 100% (DP3) abrogation of promoter activity at 24 h . Purification of nuclear protein extracts from IL-13 and IL-4-stimulated A549 cells, using a DP2 core containing affinity column, identified a 150 kDa protein under non-denaturing conditions, and two, 70 and 85 kDa proteins under denaturing conditions . These were not detectable by Coomassie blue staining in control nuclear protein extracts . Matrix assisted laser desorption ionizat